CCK-A Receptor Antagonist Research Articles

Cholecystokinin Receptor Antagonist Induces Pancreatic Stellate Cell Plasticity Rendering the Tumor Microenvironment Less Oncogenic.

CCK receptors are expressed on pancreatic cancer epithelial cells, and blockade with receptor antagonists decreases tumor growth. Activated pancreatic stellate cells or myofibroblasts have also been described to express CCK receptors, but the contribution of this novel pathway in fibrosis of the pancreatic cancer microenvironment has not been studied. We examined the effects of the nonselective CCK receptor antagonist proglumide on the activation, proliferation, collagen deposition, differential expression of genes, and migration in both murine and human PSCs. CCK receptor expression was examined using western blot analysis. Collagen production using activated PSCs was analyzed by mass spectroscopy and western blot. Migration of activated PSCs was prevented in vitro by proglumide and the CCK-B receptor antagonist, L365,260, but not by the CCK-A receptor antagonist L365,718. Proglumide effectively decreased the expression of extracellular matrix-associated genes and collagen-associated proteins in both mouse and human PSCs. Components of fibrosis, including hydroxyproline and proline levels, were significantly reduced in PSC treated with proglumide compared to controls. CCK peptide stimulated mouse and human PSC proliferation, and this effect was blocked by proglumide. These investigations demonstrate that targeting the CCK-B receptor signaling pathway with proglumide may alter the plasticity of PSC, rendering them more quiescent and leading to a decrease in fibrosis in the pancreatic cancer microenvironment.

Molecular cloning of cholecystokinin (CCK) and CCK-A receptor and mechanism of CCK-induced gastrointestinal motility in Suncus murinus

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.

Contribution of Histamine to Nociceptive Behaviors Induced by Intrathecally Administered Cholecystokinin-8.

The involvement of spinal release of histamine in the nociceptive behaviors induced by cholecystokinin-8 (CCK-8) was investigated in mice. Intrathecal (i.t.) injection of CCK-8 elicited the nociceptive behaviors consisting of biting and licking. The nociceptive behaviors induced by i.t. treatment with CCK-8 showed two bell-shaped patterns. The histamine H3 receptor antagonist significantly promoted the nociceptive behaviors induced by CCK-8 at doses of 1–100 fmol and 100 pmol. The nociceptive behaviors elicited by CCK-8 was inhibited by i.t. administration of the CCK-B receptor antagonist in a dose-dependent manner, but not by the CCK-A receptor antagonist. The nociceptive behaviors induced by CCK-8 were markedly suppressed by i.t. pretreatment with antiserum against histamine and were abolished in histidine decarboxylase-deleted gene mice. In histamine H1 receptor-deleted gene mice, the nociceptive behaviors induced at both 10 amol and 10 pmol of CCK-8 were not affected. The tachykinin neurokinin-1 (NK1) receptor antagonists inhibited CCK-8 (10 pmol)-induced nociceptive behaviors in a dose-dependent manner. CCK-8 (10 amol)-induced nociceptive behaviors was not antagonized by co-administration with the tachykinin NK1 receptor antagonists. The nociceptive behaviors elicited by CCK-8 were inhibited by i.t. administration of the antagonist for the N-methyl-D-aspartate (NMDA) receptor in a dose-dependent manner. Our results suggest that the nociceptive behaviors induced by i.t. administration of CCK-8 (10 pmol) are mediated through the spinal release of histamine and are elicited via activation of the tachykinin NK1 and NMDA receptors, whereas the nociceptive behaviors induced by i.t. administration of CCK-8 (10 amol) are mediated through the spinal release of histamine and elicited via NMDA receptor activation.

Cannabinoid CB1 Receptors Inhibit Gut-Brain Satiation Signaling in Diet-Induced Obesity.

Gut-brain signaling controls feeding behavior and energy homeostasis; however, the underlying molecular mechanisms and impact of diet-induced obesity (DIO) on these pathways are poorly defined. We tested the hypothesis that elevated endocannabinoid activity at cannabinoid CB1 receptor (CB1Rs) in the gut of mice rendered DIO by chronic access to a high fat and sucrose diet for 60 days inhibits nutrient-induced release of satiation peptides and promotes overeating. Immunoreactivity for CB1Rs was present in enteroendocrine cells in the mouse’s upper small-intestinal epithelium that produce and secrete the satiation peptide, cholecystokinin (CCK), and expression of mRNA for CB1Rs was greater in these cells when compared to non-CCK producing cells. Oral gavage of corn oil increased levels of bioactive CCK (CCK-8) in plasma from mice fed a low fat no-sucrose diet. Pretreatment with the cannabinoid receptor agonist, WIN55,212-2, blocked this response, which was reversed by co-administration with the peripherally-restricted CB1R neutral antagonist, AM6545. Furthermore, monoacylglycerol metabolic enzyme function was dysregulated in the upper small-intestinal epithelium from DIO mice, which was met with increased levels of a variety of monoacylglycerols including the endocannabinoid, 2-arachidonoyl-sn-glycerol. Corn oil failed to affect levels of CCK in DIO mouse plasma; however, pretreatment with AM6545 restored the ability for corn oil to stimulate increases in levels of CCK, which suggests that elevated endocannabinoid signaling at small intestinal CB1Rs in DIO mice inhibits nutrient-induced CCK release. Moreover, the hypophagic effect of AM6545 in DIO mice was reversed by co-administration with the CCKA receptor antagonist, devazepide. Collectively, these results provide evidence that hyperphagia associated with DIO is driven by a mechanism that includes CB1R-mediated inhibition of gut-brain satiation signaling.

Effects of Cholecystokinin (CCK) on Gut Motility in the Stomachless Fish Ballan Wrasse (Labrus bergylta).

Cholecystokinin (CCK) is well-known as a key hormone that inhibits stomach emptying and stimulates midgut motility in gastric species. However, the function of CCK related to gut motility in agastric fish, especially in fish with a short digestive tract such as ballan wrasse, remains unknown. Here we present a detailed description of the spatio-temporal quantification of intestinal motility activity in vitro comprising the complete intestinal tract in ballan wrasse. We show that CCK modulates intestinal motility, having multiple effects on motility patterns depending on location in the gut and types of contractions. CCK reduced propagating contractions in the foregut, but it increased both non-propagating and propagating contractions in the hindgut. CCK also altered the direction of propagating contractions, as it reduced anterograde ripples and slow propagating contractions. The velocity of propagating contractions was slowed down by CCK. CCK also reduced the amplitude of standing contractions and ripples, but it did not alter the amplitude of slow propagating contractions. The presence of CCKA receptor antagonist modulated the motility responses of ballan wrasse intestines when exposed to CCK. We also showed that CCK reduced the intestinal length and stimulated motility to empty the gallbladder. Based on our findings we hypothesize that CCK, mainly through the CCKA receptor, modulates non-propagating and propagating contractions to optimize digestion and absorption and regulate the intestinal evacuation in ballan wrasse. We also found evidence that the modulation of intestinal motility by CCK is different in agastric fish from that in gastric vertebrates. We suggest that this is an evolutionary adaptation to optimize digestion without a stomach.

Intragastric nutrient infusion reduces motivation for food in male and female rats.

The idea that gut-derived satiation signals influence food reward has recently gained traction, but this hypothesis is largely based on studies focused on neural circuitry, not the peripherally released signals. Here, we directly tested the hypothesis that intragastric (IG) nutrient infusion can suppress motivation for food. In a series of experiments, IG sucrose infusion (15 kcal) significantly and reliably reduced operant responding for a sucrose reward on a progressive ratio (PR) schedule. Moreover, food deprivation for 24 h before the test session did not prevent the suppressive effect of nutrients. The suppressive effect of IG sucrose on fixed ratio 5 (FR5) operant responding was also assessed as a comparison. The effect of IG nutrients to reduce motivation was not limited to sucrose; IG Ensure infusion (9.3 kcal) also significantly reduced PR operant responding for sucrose pellets. To verify that these effects were not secondary to the osmotic challenge of concentrated nutrients, we tested IG infusion of noncaloric saline solutions equiosmolar to 40% sucrose or Ensure and found no effect. Finally, we focused on glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) as candidate mediators for the effect of IG nutrients. Pretreatment with exendin-9, a GLP-1 receptor antagonist, delivered intraperitoneally, significantly attenuated the ability of IG nutrients to suppress PR responding and breakpoint in males, but not in females, whereas pretreatment with devazepide, a CCKA receptor antagonist, failed to do so in both sexes. Together, these data support the idea that nutrient-induced satiation signals influence food reward and may implicate GLP-1 in this process.

Presynaptically mediated effects of cholecystokinin-8 on the excitability of area postrema neurons in rat brain slices

Cholecystokinin (CCK) is a well-known gut hormone that shows anorexigenic effects via action at peripheral and central receptors. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural elements unique access to circulating substances. Immunohistochemical studies show CCK-A receptors in the area postrema, and we reported CCK-sensitive area postrema neurons. However, the receptive mechanism of CCK in area postrema neurons still remains unexplained. We investigated the responses of area postrema neurons to agonists and antagonists of CCK receptors using whole cell and perforated patch-clamp recordings in rat brain slices. The application of CCK-8 elicited excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents), a shift toward larger amplitude mEPSCs, and increases in the frequency of action potentials. These changes were found mostly in cells not displaying the hyperpolarization-activated cation current (Ih), except for small excitatory changes in a minority of Ih-positive neurons. Tonic inward currents or an inhibitory response to CCK-8 were never seen. Analysis of the amplitude of mEPSCs before and after the administration of CCK-8 indicated the responses mediated via the presynaptic receptors. The effect of CCK-8 was abolished in the presence of CNQX (AMPA type glutamate receptor antagonist). In the presence of lorglumide (a selective CCK-A receptor antagonist), CCK-8-induced excitatory responses were inhibited. No cells responded to the administration of non-sulfated CCK-8 (CCK-8NS, a selective CCK-B receptor agonist). We conclude that CCK-8 exerts its action via presynaptic CCK-A receptors to facilitate glutamate release onto Ih-negative area postrema cells.

Cholecystokinin mediates progression and metastasis of pancreatic cancer associated with dietary fat.

Obesity and dietary fat are associated with increased risk of several malignancies including pancreatic cancer. The incidence of pancreatic cancer is increased in countries that consume diets high in fat. The purpose of this study was to assess the relationship and mechanism of action between dietary fat and endogenous cholecystokinin (CCK) on pancreatic tumor growth and metastasis in an immunocompetent animal model. C57BL/6 mice were placed on regular, low-fat, or high-fat diets for 8weeks before establishment of Panc-02 orthotopic pancreatic tumors. Mice were then treated with a CCK-A receptor antagonist, devazepide, or vehicle for an additional 2.5weeks. Pancreas tumors were weighed and metastases counted. Blood CCK levels were measured by radioimmunoassay (RIA). Tissues were examined histologically and studied for genes associated with metastasis by RT-PCR array. Effects of the CCK antagonist on Panc-02 cells invasiveness was assessed in a Matrigel invasion assay. Mice that received the high-fat diet had larger tumors and tenfold higher serum CCK levels by RIA compared to normal diet controls (p<0.01). Pancreatic tumors in high-fat diet mice treated with the antagonist had fewer intravascular tumor emboli and metastases compared to controls. The reduction in tumor emboli correlated with decreased vascular endothelial growth factor-A (VEGF-A) expression in tumors (p<6×10(-9)). In vitro invasiveness of Panc-02 cells also was reduced by CCK-A receptor antagonist treatment (p=1.33×10(-6)). CCK is a mediator of dietary fat-associated pancreatic cancer. CCK is also involved in the invasiveness of pancreatic tumors through a mechanism involving VEGF-A.

Duodenal Lipid Sensing Activates Vagal Afferents to Regulate Non-Shivering Brown Fat Thermogenesis in Rats

Previous evidence indicates that duodenal lipid sensing engages gut-brain neurocircuits to determine food intake and hepatic glucose production, but a potential role for gut-brain communication in the control of energy expenditure remains to be determined. Here, we tested the hypothesis that duodenal lipid sensing activates a gut–brain–brown adipose tissue neuraxis to regulate thermogenesis. We demonstrate that direct administration of lipids into the duodenum increases brown fat temperature. Co-infusion of the local anesthetic tetracaine with duodenal lipids abolished the lipid-induced increase in brown fat temperature. Systemic administration of the CCKA receptor antagonist devazepide blocked the ability of duodenal lipids to increase brown fat thermogenesis. Parenchymal administration of the N-methyl-d-aspartate receptor blocker MK-801 directly into the caudomedial nucleus of the solitary tract also abolished duodenal lipid-induced activation of brown fat thermogenesis. These findings establish that duodenal lipid sensing activates a gut–brain–brown fat axis to determine brown fat temperature, and thereby reveal a previously unappreciated pathway that regulates thermogenesis.

Behavioral and cortical EEG evaluations confirm the roles of both CCKA and CCKB receptors in mouse CCK-induced anxiety

This study investigated the roles of cholecystokinin (CCK)A and CCKB receptors on CCK-4-induced anxiety-like behaviors in mice through behavioral and neural evaluations. Anxiety-like behaviors in mice were induced by an intracerebroventricular (i.c.v.) administration of CCK-4, which can bind to both CCKA and CCKB receptors. The effects of CCKA and CCKB receptor antagonists (devazepide and CI-988, respectively) were examined using mouse anxiety tests (elevated-plus maze and light–dark box) and also by examining neuronal activities through EEG monitoring and c-Fos immunohistochemistry in the cortex and amygdala. CCK-4 (3μg/kg of body weight i.c.v.) significantly induced mouse anxiety-like behaviors in the anxiety tests and also affected their EEG patterns with respect to pre-drug tracing, resulting in increase in spectral power in relative power distribution in the delta and theta bands (0.5–5Hz frequency bands) and also in increase in c-Fos immunopositive neuron counts. These CCK-4 effects were completely suppressed by 1.0mg/kg CCKB receptor antagonist, CI-988, while the same amount of CCKA receptor antagonist, devazepide was partly able to suppress the same effects. These findings indicated that not only CCKB receptors but also CCKA receptors in the brain play important roles in regulating anxiety-like behaviors in mice. The present study also proposed a possibility that cortical EEG is useful for assessing anxiety.

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

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

Differential stimulation of duodenal HCO3− secretion by luminal fatty acids

Luminal nutrients enhance mucosal defenses via gut hormone release. We examined the effects of long‐chain (LCFA) or short‐chain fatty acid (SCFA) receptor agonists on duodenal HCO3− secretion (DBS). We measured DBS with pH and CO2 electrodes in a perfused duodenal loop in anesthetized rats. Acetate or propionate, GPR43 agonist phenylacetamide 1 (PA1) or GPR40 agonist GW9508 was luminally perfused +/− iv dipeptidyl peptidase 4 inhibitor NVP728 or hormone receptor antagonist. LCFA receptor GPR40 and SCFA receptors GPR41/43 were expressed in the duodenal enteroendocrine cells. Luminal perfusion of acetate or propionate similarly increased DBS, enhanced by NVP728, while increasing GLP‐2 release in portal blood. Acetate‐induced DBS was reduced by the monocarboxylate transporter inhibitor, suggesting the acetate absorption‐mediated and GLP‐2‐mediated DBS. PA1 dose‐dependently increased DBS, unaffected by NVP728 or CCK‐A receptor antagonist, but inhibited by atropine or 5‐HT4 antagonist. The GPR40 agonist GW9508 increased DBS with GLP‐2 release, enhanced by NVP728. Activation of GPR40 by LCFA and possibly GPR41 by SCFA increases DBS via GLP‐2 release, whereas GPR43 activation stimulates DBS via muscarinic and 5‐HT4 receptor activation. The presence of fatty acid sensing in the duodenal mucosal defenses may be implicated in the pathogenesis of functional dyspepsia syndromes.VA Merit Review, R01 DK54221

Duodenal PKC-δ and Cholecystokinin Signaling Axis Regulates Glucose Production

OBJECTIVEMetabolism of long-chain fatty acids within the duodenum leads to the activation of duodenal mucosal protein kinase C (PKC)-δ and the cholecystokinin (CCK)-A receptor to lower glucose production through a neuronal network. However, the interfunctional relationship between duodenal PKC-δ and CCK remains elusive. Although long-chain fatty acids activate PKC to stimulate the release of CCK in CCK-secreting cells, CCK has also been found to activate PKC-δ in pancreatic acinar cells. We here evaluate whether activation of duodenal mucosal PKC-δ lies upstream (and/or downstream) of CCK signaling to lower glucose production.RESEARCH DESIGN AND METHODSWe first determined with immunofluorescence whether PKC-δ and CCK were colocalized within the duodenal mucosa. We then performed gain- and loss-of-function experiments targeting duodenal PKC-δ and the CCK-A receptor and evaluated the impact on changes in glucose kinetics during pancreatic (basal insulin) clamps in rats in vivo.RESULTSImmunostaining of PKC-δ was found to colocalize with CCK in the duodenal mucosa. Intraduodenal coinfusion of either the CCK-A receptor antagonist MK-329 or CR-1409 with the PKC activator negated the ability of duodenal mucosal PKC-δ activation to lower glucose production during the pancreatic clamps in normal rats. Conversely, molecular and pharmacological inhibition of duodenal PKC-δ did not negate the ability of the duodenal CCK-A receptor agonist CCK-8 to lower glucose production, indicating that activation of duodenal PKC-δ lies upstream (and not downstream) of CCK signaling. Finally, intraduodenal PKC activator infusion failed to lower glucose production in rats with high-fat diet–induced duodenal CCK resistance.CONCLUSIONSIn summary, activation of duodenal PKC-δ leads to the stimulation of CCK release and activation of the CCK-A receptor signaling axis to lower glucose production in normal rats, but fails to bypass duodenal CCK-resistance in high fat-fed rats.

L-364,718 Potentiates Electroacupuncture Analgesia Through Cck-A Receptor of Pain-Related Neurons in the Nucleus Parafascicularis

Electroacupuncture (EA) has been successfully used to alleviate pain produced by various noxious stimulus. Cholecystokinin-8 (CCK-8) is a neuropeptide involved in the mediation of pain. We have previously shown that CCK-8 could antagonize the analgesic effects of EA on pain-excited neurons (PENs) and pain-inhibited neurons (PINs) in the nucleus parafascicularis (nPf). However, its mechanism of action is not clear. In the present study, we applied behavioral and neuroelectrophysiological methods to determine whether the mechanisms of CCK-8 antagonism to EA analgesia are mediated through the CCK-A receptors of PENs and PINs in the nPf of rats. We found that focusing radiant heat on the tail of rats caused a simultaneous increase in the evoked discharge of PENs or a decrease in the evoked discharge of PINs in the nPf and the tail-flick reflex. This showed that radiant heat could induce pain. EA stimulation at the bilateral ST 36 acupoints in rats for 15 min resulted in an inhibition of the electrical activity of PEN, potentiation of the electrical activity of PIN, and prolongation in tail-flick latency (TFL), i.e. EA stimulation produced an analgesic effect. The analgesic effect of EA was antagonized when CCK-8 was injected into the intracerebral ventricle of rats. The antagonistic effect of CCK-8 on EA analgesia was reversed by an injection of CCK-A receptor antagonist L-364,718 (100 ng/μl) into the nPf of rats. Our results suggest that the pain-related neurons in the nPf have an important role in mediating EA analgesia. L-364,718 potentiates EA analgesia through the CCK-A receptor of PENs and PINs in the nPf.

ChemInform Abstract: Dual CCK-A and CCK-B Receptor Antagonists. Part 2. Preparation and Structure Activity Relationships of 5-Alkyl-9-methyl-1,4-benzodiazepines and Discovery of FR208419.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synergistic effect of CART (cocaine- and amphetamine-regulated transcript) peptide and cholecystokinin on food intake regulation in lean mice

BackgroundCART (cocaine- and amphetamine-regulated transcript) peptide and cholecystokinin (CCK) are neuromodulators involved in feeding behavior. This study is based on previously found synergistic effect of leptin and CCK on food intake and our hypothesis on a co-operation of the CART peptide and CCK in food intake regulation and Fos activation in their common targets, the nucleus tractus solitarii of the brainstem (NTS), the paraventricular nucleus (PVN), and the dorsomedial nucleus (DMH) of the hypothalamus.ResultsIn fasted C57BL/6 mice, the anorexigenic effect of CART(61-102) in the doses of 0.1 or 0.5 μg/mouse was significantly enhanced by low doses of CCK-8 of 0.4 or 4 μg/kg, while 1 mg/kg dose of CCK-A receptor antagonist devazepide blocked the effect of CART(61-102) on food intake. After simultaneous administration of 0.1 μg/mouse CART(61-102) and of 4 μg/kg of CCK-8, the number of Fos-positive neurons in NTS, PVN, and DMH was significantly higher than after administration of each particular peptide. Besides, CART(61-102) and CCK-8 showed an additive effect on inhibition of the locomotor activity of mice in an open field test.ConclusionThe synergistic and long-lasting effect of the CART peptide and CCK on food intake and their additive effect on Fos immunoreactivity in their common targets suggest a co-operative action of CART peptide and CCK which could be related to synergistic effect of leptin on CCK satiety.

Responses of human sling and clasp fibers to cholecystokinin (CCK) and gastrin through CCK receptors

Cholecystokinin (CCK) and gastrin exert their influences via CCK receptors. This research was conducted to look at the responses of the sling and clasp fibers forming the human lower esophageal sphincter (LES) to CCK and gastrin, and the role of CCK receptors in the responses. Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing subtotal esophagectomy. Isometric tension responses of the strips to CCK-8 and gastrin-17 were studied, and the maximum effect (E(max)) for each agonist was derived. CCK-A receptor antagonist, CR1409 and CCK-B antagonist, CR2945 were applied to sling and clasp fibers and their pK(B) values were calculated. Sling fibers produced significant contractions following exposure to CCK-8 and gastrin-17, while clasp fibers had less responses to the two agents. CR1409 and CR2945 inhibited responses of sling to CCK-8 in a concentration-dependent fashion. The inhibition effects of the two antagonists on clasp fibers were not measurable because there was a mild contraction of the fiber in response to CCK-8. The contractions generated by sling fibers following exposure to CCK and gastrin are greater than that produced by clasp fibers. CCK-A receptors are more important for the generation of contractions by the sling fibers, whereas both CCK-A and CCK-B receptors are involved in the functional regulation of the clasp fibers. [Corrections added after online publication 28 April 2008: in the Background and Aims section of the preceding abstract, all instances of 'CKK' were corrected to 'CCK'. In the final sentence of the abstract 'CCKA'was corrected to 'CCK-A'. In the article title '(CKK)' was corrected to '(CCK)'.].

The role of protein kinase C-mediated phosphorylation of type III inositol 1, 4, 5-triphosphate receptor in cholecystokinin octapeptide induced calcium mobilization in gastric antral smooth muscle cells

To study the effects of sulfated cholecystokinin octapeptide (sCCK-8S) on intracellular calcium release and extracellular calcium influx in gastric antral smooth muscle cells (SMC) and the mechanism thereof. (1) Longitudinal muscle (LM) and circular muscle (CM) strips of gastric antrum and pylorus were isolated from SD rats and suspended in a tissue chamber to record the contractile responses by polyphysiography. (2) Immunoprecipitation, electrophoresis, and immunoblotting were used to detect the phosphorylation of type III inositol 1, 4, 5-triphosphate receptor (InsP(3)R(3)) in the SMCs. (3) The responsiveness of gastric SMC to CCK-8S was examined by using fura-2-loaded microfluorimetric measurement of intracellular calcium concentration ([Ca(2+)]i). (4) The current of L-type calcium channels (ICaL) was recorded by using patch-clamp techniques. (1) Significant changes to CCK-8S were found in the mean contractile amplitude of the CM and frequency of LM of gastric antrum and could be suppressed by CCK-A receptor (CCK-AR) antagonist and ATPase inhibitors. (2) CCK-8S stimulation of SMC resulted in PKC-dependent phosphorylation of the InsP(3)R(3). (3) CCK-8S-evoked significant increase in [Ca(2+)]i [from (69 +/- 7) mol/L to (472 +/- 36) nmol/L, P < 0.01] could be suppressed by CCK-AR antagonist, ATPase inhibitors and protein kinase C (PKC) activator; whereas on condition that extracellular calcium was removed or L-type calcium inhibitor nifedipine was added a small but significant increase of [Ca(2+)]i could be still elicited by CCK-8S. (4) CCK-8S-intensified calcium current [from (-56 +/- 7) pA to (-89 +/- 6) pA, P < 0.01] could be apparently inhibited by respective administration of nifedipine, ATPase inhibitors, and calcium dependent chloride channel (I(Cl-Ca)) blocker (all P < 0.01). CCK-8S-evoked [Ca(2+)]i increase in gastric antral SMCs depends on the release of intracellular calcium stores, which is regulated by PKC mediated phosphorylation of InsP(3)R(3). The released intracellular calcium in turn activates the L-type voltage-dependent calcium channels (VDCC) through the activation of calcium dependent chloride channels, and ultimately results in the occurrence of contraction response of smooth muscles.

Pentagastrin-induced protein synthesis in the parotid gland of the anaesthetized rat, and its dependence on CCK-A and -B receptors and nitric oxide generation

In parotid glands of pentobarbitone-anaesthetized rats, the incorporation of [3H]leucine into trichloroacetic acid-insoluble materials, reflecting protein synthesis, increased by 17% (compared to saline-treated rats) in response to infusion of pentagastrin (20 microg kg(-1), i.v. for 1 h) under muscarinic and alpha- and beta-adrenoceptor blockade. Both the CCK-A receptor antagonist lorglumide (48 mg kg(-1), i.v.) and the CCK-B receptor antagonist itriglumide (5.5 mg kg(-1), i.v.), given separately, prevented the expected increase in pentagastrin and, in addition, reduced the glandular protein synthesis by 16 and 12%, respectively, below the level of saline-treated rats. In rats treated with saline only, the glandular protein synthesis was reduced by 22% by the CCK-A receptor antagonist and by 17% by the CCK-B receptor antagonist; combined, the two antagonists caused no further reduction (20%). There was no increase in the glandular protein synthesis of pentagastrin-treated rats compared to that of the saline-treated rats when both groups of rats were exposed to a combination of the two types of CCK receptor antagonists. In pentagastrin-treated rats, the protein synthesis in the parotid glands was 23% less in the presence of the non-selective nitric oxide (NO) synthase inhibitor L-NAME (30 mg kg(-1), i.v.) than in its absence; the result was the same (23%) when the neuronal NO synthase inhibitor Nomega-propyl-L-arginine (N-PLA; 30 mg kg(-1), i.v.) replaced L-NAME. The protein synthesis in rats treated with saline only was not reduced by L-NAME; nor was the protein synthesis of saline-treated rats different from that of pentagastrin- and L-NAME-treated rats. Thus, under 'basal' conditions, a portion of the glandular protein synthesis, as well as the whole increase in synthesis in response to administration of pentagastrin, engaged both types of CCK receptors. Furthermore, NO generation, owing to neuronal NO synthase activity, was required for the increase to occur in response to pentagastrin, whereas a non-NO-dependent pathway was responsible for the protein synthesis under 'basal' conditions.

Effect of cholecystokinin octapeptide on diacylglycerol-PKC signaling pathway in rat pulmonary interstitial macrophages stimulated by lipopolysaccharide1

To investigate the effect of cholecystokinin octapeptide (CCK-8) on the diacylglycerol-protein kinase C (DAG-PKC) signaling pathway in rat pulmonary interstitial macrophages (PIM) stimulated by lipopolysaccaride (LPS). The PIM from rat lung tissues were isolated using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. DAG content and PKC activity were measured by radioenzymatic assay. The translocation of PKCzeta was determined by semi-quantitative immunoblot analysis. CCK-8, at high concentrations (1 x 10(-6) - 1 x 10(-5) mol/L), decreased DAG content and inhibited PKC activity and PKCzeta translocation compared with that in rat resting PIM of a control group (P< 0.01). LPS increased DAG content, and promoted PKC activity and PKCzeta translocation (P< 0.01). CCK-8 decreased LPS-induced DAG content and inhibited LPS-induced PKC activity and PKCzeta translocation significantly at 1 x 10(-8) - 1 x 10(-5) mol/L (P< 0.01). This inhibitory effect of CCK-8 could be abrogated partly by proglumide (non-selective CCK receptor antagonist), CR-1409 (selective CCK-A receptor antagonist), and CR-2945 (selective CCK-B receptor antagonist) in a concentration-dependent manner (P< 0.01). CCK-8 was a negative modulator of the DAG-PKC signaling pathway in rat resting PIM, which is very important for maintaining body homeostasis. It significantly inhibited LPS-induced DAG content, PKC activity and PKCzeta translocation in a concentration-dependent manner. The CCK receptor, especially the CCK-A receptor, might play a major role in this process.