OBJECTIVE

Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer.

METHODS

The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy.

RESULTS

Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001).

CONCLUSIONS

These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

Radioresistance is one of the major barriers to improve the survival rate of breast cancer patients. Cyclooxygenase 2 (COX-2) is usually overexpressed in highly invasive and metastatic breast cancer, which may indicate an association with breast cancer radioresistance. The function role of COX-2 was investigated by using a radioresistant breast cancer cell line MDA-MB-231/RR10 and its parental cell line MDA-MB-231 cells before or after COX-2 was silenced by a specific small hairpin RNA (shRNA). The cell proliferation, migration, invasion, colony formation, and apoptosis were measured by CCK-8, scratch-wound, transwell, clone formation assay, and flow cytometry. Protein and mRNA expression were analyzed by Western blot and quantitative reverse transcriptase-polymerase chain reaction. COX-2 is upregulated in MDA-MB-231/RR10 cells compared with in MDA-MB-231 cells, and silencing of COX-2 expression by shRNA in MDA-MB-231/RR10 cells decreases the expression of Bcl-2 and Bcl-XL, but increases the proapoptotic protein BAK, leading to the increased apoptosis following treatment with γ-irradiation in comparison with those in control cells. Silencing of COX-2 also increases the expression of β-catenin and E-cadherin, two anti-invasion proteins, resulting in reduced cell migration and invasion tested by transwell chambers and wound-healing assays. Further study demonstrated that COX-2-induced radioresistance is negatively regulated through the phosphorylation of p38 at Tyr182, and that the phosphorylation of p38 induced by TNF-alpha reduces the expression of Bcl-2, BCL-XL, but increases β-catenin and E-cadherin, leading to the decreased invasiveness of cells. Our data suggest that COX-2, p38, Bcl-2, Bcl-XL, β-catenin, and E-cadherin may be considered as potential therapeutic targets against radioresistant breast cancer.

We have very recently demonstrated the low acidity of gastric juice and the high susceptibility to the development of gastric ulceration in Otsuka Long-Evans Tokushima Fatty (OLETF) rats not expressing CCK-A receptors. In the present study, gastric emptying in this strain was examined and compared with control Long-Evans Tokushima Otsuka (LETO) rats. Gastric emptying was evaluated by the phenol red method. Gastric emptying 30 and 60 min after a liquid meal in OLETF rats was significantly delayed compared to that in control LETO rats. Intraperitoneal injection of CCK-8 at a dose of 5 microg/kg significantly inhibited gastric emptying in control LETO rats, whereas the same dose of CCK-8 failed to inhibit gastric emptying in OLETF rats. These results suggest for the first time that gastric emptying was suppressed in OLETF rats. We also confirmed with this mutant that CCK delays gastric emptying through the CCK-A receptors.

BACKGROUND

The role of cholecystokinin (CCK) and gastrin in the development and growth of pancreatic cancer cells is controversial. The aim of this study was to evaluate the role of CCK-8S, gastrin-17, bombesin, and their antagonists on cell lines from patients with pancreatic cancer.

METHODS

Cell lines were established from pancreatic cancers operated on at our department. The cells were grown in 10% fetal calf serum (FCS). The effects of CCK-8S, gastrin-17, bombesin, and their antagonists in different concentrations and for different time intervals were studied. The cell number was evaluated with the XTT method.

RESULTS

The cell line LN 36 responded with increased cell number to stimulation by gastrin-17 and decreased cell number to inhibition by the CCK-B receptor antagonist L-365,260. In contrast, LPC 1 responded with increased cell number to CCK-8S and decreased cell number to the CCK-A receptor antagonist devazepide. LPC 2, 6, and 7 were stimulated by CCK-8S, gastrin-17, and their antagonists. LPC 3 showed decreased cell number after inhibition by the antagonists, and LPC 5 and 10 showed increased cell number after stimulation by CCK-8S and gastrin-17. LPC 4 was stimulated by CCK-8S, and LPC 8 was stimulated by all substances except gastrin-17. Intermittent administration of the substances to LN 36 led to a greater effect on the cell number than administration every day, which was not the case with LPC 1 and LPC 3. Bombesin led to an increased growth in LPC 5 but not in LPC 3.

CONCLUSIONS

CCK-8S and gastrin-17 led to an increased cell number in some cell lines. A blockade of the CCK-A and CCK-B receptors by their antagonists led to an increased, an unaffected, or a decreased cell number of the cell lines. The effect of bombesin on different cell lines also varied. This shows a great heterogenicity among pancreatic cancer cells from different patients.

In the rat model, polyunsaturated vegetable oil has been shown to stimulate pancreatic secretion and promote pancreatic carcinogenesis, whereas dietary fish oil has been found to protect against carcinogenesis. Because cholecystokinin (CCK), a hormonal polypeptide secreted from the upper small intestine after food stimulation, is the most important known humoral stimulus of pancreatic secretion and also because this gut hormone has been shown to promote pancreatic carcinogenesis in the rat, we decided to study the effects of various triglycerides on CCK secretion in this species. Small amounts (2.5 mL) of corn oil, beef tallow, fish oil, medium-chain triglyceride (MCT) oil or saline (control) were administered to groups of five fasted rats. Plasma CCK levels were measured using a specific and sensitive radioimmunoassay. The maximal CCK increments for corn oil, beef tallow, fish oil and MCT oil were 3.0 +/- 0.5, 2.1 +/- 0.6 and 7.2 +/- 0.4 pmol/L, respectively. All the increments were significantly greater (p less than 0.05) than the change found after saline administration (-0.8 +/- 0.3 pmol/L). In another experiment, plasma CCK levels after intragastric administration of MCT oil reached a peak increment of 6.4 +/- 0.4 pmol/L after 240 min and continued to be significantly increased for the entire 480-min study period. It was concluded that all four triglycerides caused a significant CCK release in the rat and that the MCT oil was the most powerful stimulator of CCK secretion among the triglycerides studied.

This present study was designed to investigate the effects of alpha-1-antitrypsin (AAT) on oxidative stress in preeclampsia (PE) by regulating p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. HTR8/SVneo cells were randomly assigned into normal, hypoxia/reoxygenation (H/R), HR + AAT and HR + siRNA-AAT groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the mRNA and protein expressions of p-p38MAPK, AAT, signal transducer and activator of transcription 1 (STAT1) and activating transcription factor2 (ATF2). Flow cytometry, scratch test, cell counting kit-8 (CCK-8) assay and the 3-(4,5)-dimethylthiazol (-z-y1)-3,5-di- phenyltetrazolium bromide (MTT) assay were conducted to detect reactive oxygen species (ROS) and cell apoptosis, cell migration, proliferation and cytotoxicity, respectively. Mouse models in PE were established, which were divided into normal pregnancy (NP), PE and PE + AAT groups with blood pressure and urine protein measured. Chromatin immunoprecipitation (ChIP) and enzyme-linked immunosorbent assay (ELISA) were conducted to detect the activity of oxidative stress-related kinases and expressions of inflammatory cytokines and coagulation-related factors in cells and mice placenta. Immunohistochemistry, Western blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect AAT and p38MAPK expressions, apoptosis-related protein expressions, and apoptosis rate in mice placenta. Compared with the normal group, the H/R group had decreased expression of AAT, activity of superoxide dismutase (SOD) and GSH-Px, cell proliferation and migration, but increased p38MAPK, STAT1, ATF2, MDA, H2O2, inflammatory cytokines, coagulation-related factors, cell cytotoxicity, ROS, apoptotic factors and apoptosis rate. Compared with the H/R group, the HR + ATT group had increased expressions of AAT, activity of SOD and GSH-Px, cell proliferation and migration but decreased p38MAPK, STAT1, ATF2, malonyldialdehyde (MDA), H2O2, inflammatory cytokines and coagulation-related factors, cell cytotoxicity, ROS, apoptotic factors and apoptosis rate, while opposite results were observed in the HR + siRNA-ATT group. Compared with the NP group, the PE group had decreased activity of SOD and GSH-Px but increased MDA, H2O2, AAT, p38MAPK, inflammatory cytokines, coagulation-related factors and apoptosis rate. The indexes in the PE + AAT group were between the NP and PE groups. Thus, we concluded that AAT suppressed oxidative stress in PE by inhibiting p38MAPK signaling pathway.

The gray garden slug, Deroceras reticulatum (Gastropoda: Pulmonata), is one of the most common terrestrial molluscs. Research for this slug has focused mainly on its ecology, biology, and management due to the severe damage it causes on a wide range of vegetables and field crops. However, little is known about neuropeptides and hormonal signalings. This study, therefore, aimed to establish the transcriptome of D. reticulatum and to identify a comprehensive repertoire of neuropeptides in this slug. Illumina high-throughput sequencing of the whole body transcriptome of D. reticulatum generated a total of 5.9 billion raw paired-end reads. De novo assembly by Trinity resulted in 143,575 transcripts and further filtration selected 120,553 unigenes. Gene Ontology (GO) terms were assigned to 30,588 unigenes, composed of biological processes (36.9%), cellular components (30.2%) and molecular functions (32.9%). Functional annotation by BLASTx revealed 39,987 unigenes with hits, which were further categorized into important functional groups based on sequence abundance. Neuropeptides, ion channels, ribosomal proteins, G protein-coupled receptors, detoxification, immunity and cytoskeleton-related sequences were dominant among the transcripts. BLAST searches and PCR amplification were used to identify 65 putative neuropeptide precursor genes from the D. reticulatum transcriptome, which include achatin, AKH, allatostatin A, B and C, allatotropin, APGWamide, CCAP, cerebrin, conopressin, cysteine-knot protein hormones (bursicon alpha/beta and GPA2/GPB5), elevenin, FCAP, FFamide, FVamide (enterin, fulicin, MIP and PRQFVamide), GGNG, GnRH, insulin, NdWFamide, NKY, PKYMDT, PRXamide (myomodulin, pleurin and sCAP), RFamide (CCK/SK, FMRFamide, FxRIamide, LFRFamide, luqin and NPF), and tachykinin. Over 330 putative peptides were encoded by these precursors. Comparative analysis among different molluscan species clearly revealed that, while D. reticulatum neuropeptide sequences are conserved in Mollusca, there are also some unique features distinct from other members of this species. This is the first transcriptome-wide report of neuropeptides in terrestrial slugs. Our results provide comprehensive transcriptome data of the gray garden slug, with a more detailed focus on the rich repertoire of putative neuropeptide sequences, laying the foundation for molecular studies in this terrestrial slug pest.

OBJECTIVE

Hypoxia may play a role in the survival of ectopic endometrial cells. This study aimed to explore how hypoxia responsive miR-210 is involved in cell survival and autophagic response of endometriotic cells.

METHODS

The expression of hypoxia-inducible factor 1-alpha (HIF-1α) and miR-210 in eutopic and ectopic endometrial tissues were measured. The expression changes of HIF-1α and miR-210 in ovarian endometriotic cell line CRL-7566 after hypoxic culture were further explored. The influence of miR-210 on cell viability and apoptosis was quantified using CCK-8 assay and flow cytometry analysis. The effect of miR-210 on Bcl-2 expression and the effect of miR-210/Bcl-2 axis on autophagy in the cells were measured by Western blot analysis.

RESULTS

Ectopic lesion had stronger HIF-1α positive signals, as well as more HIF-1α positive cells per visual field than the eutopic endometrium. MiR-210 expression was also elevated in the ectopic lesions. In in-vitro models, CRL-7566 cells had significantly higher expression of HIF-1α and miR-210 after hypoxic treatment. MiR-210 overexpression partly preserved cell viability in hypoxia, while miR-210 knockdown facilitated the loss of cell viability. In addition, miR-210 significantly attenuated hypoxia-induced apoptosis in CRL-7566 cells. Enforced miR-210 overexpression significantly promoted autophagy in hypoxia. Knockdown of endogenous Bcl-2 significantly enhanced autophagy, the effect of which was similar to that of miR-210.

CONCLUSIONS

The hypoxia-induced higher miR-210 expression may contribute to pathological development of endometriosis at least through enhancing cell survival and promoting autophagy via Bcl2/Beclin-1 axis.

The ability of the cholecystokinin (CCK) receptor antagonists, MK-329 and L-365,260, to selectively inhibit 125I-Bolton-Hunter-CCKCCK binding sites accumulating proximal to ligatures on the cervical vagus. Incubation of nerve sections in the presence of both antagonists produced an additive effect, indicating that both CCK-A and CCK-B binding sites are transported towards the periphery. In contrast, CCK binding sites accumulating distal to the ligature possessed the pharmacological characteristics of the CCK-B receptor sub-type only.

Bombesin's influence on gastric vagal afferent discharge (GVAD) was studied in urethan-anesthetized rats. Vehicle and peptides were injected intravenously at 30-min intervals. Cholecystokinin (CCK; 300 pmol) and bombesin (300 pmol) increased ongoing multiunit GVAD by 153 +/- 59 and 162 +/- 37%, respectively; similar increases were induced by a second injection of bombesin and CCK. The bombesin antagonist, ICI-216140, prevented bombesin-induced increase in GVAD, whereas the CCK response was not influenced. The CCK-A receptor antagonist devazepide reduced the activation of GVAD induced by bombesin from 107 +/- 11 to 63 +/- 6%, while abolishing the CCK response. Devazepide given alone or in combination with ICI-216140 did not modify gastric distension (3 ml)-induced increase in GVAD. Of 22 single units that were activated by gastric load (4 ml), 17 and 20 units responded also to bombesin (620 pmol) and CCK (870 pmol), respectively. Of the nine units that did not respond to gastric load, eight had an increase in GVAD induced by both bombesin and CCK. There was no specific binding of 125I-labeled [Tyr4]bombesin on cervical vagus, either intact or 24 h after ligation. These data suggest that intravenous bombesin-induced stimulation of GVAD is indirect and initially mediated through specific receptor activation influencing gastric smooth muscle and the release of CCK.

Postprandial cholecystokinin (CCK) has been suggested as an important mediator of disruption of migrating myoelectric complexes (MMC) after a meal. However, the role of CCK in regulating small intestinal motility in rats and the participation of central and/or peripheral CCK-A and B receptors in CCK actions, are still unclear. For this study, Sprague-Dawley rats were prepared with electrodes in the small intestine, a catheter in the jugular vein and an intracerebroventricular (i.c.v.) cannula. Postprandial disruption of the MMC was blocked by the i.v. infusion of the CCK-B antagonist L-365,260 (2 x 10(-7) mol/kg), but not by the infusion of the CCK-A antagonist L-364,718. When administered i.c.v., L-364,718 (2.25 x 10(-9) mol), but not L-365,260, restored the MMC pattern. The i.v. infusion of CCK-8 (1-3 x 10(-9) mol/kg) or CCK-4 (10(-7) mol/kg) disrupted the MMC pattern. CCK-8 effects where prevented by the i.v. infusion of either L-364,718 or L-365,260. Administered i.c.v., only the antagonist L-364,718 prevented CCK-8 disruption of the MMC. These results suggest that CCK-mediated motor changes after a meal are due to stimulation of peripheral CCK-B receptors. CCK also induces a release of central CCK that through CCK-A receptors participates on MMC disruption.

Cholecystokinin octapeptide (CCK-8) is a potent corticotroph secretagogue. Consistent with earlier reports, the present results demonstrate that CCK-8 administration to rats elevates circulating beta-endorphin and adrenocorticotropin, but not alpha-melanocyte-stimulating hormone concentrations. This response was blocked by dexamethasone pretreatment, but not by vagotomy, and it could not be reproduced by i.c.v. CCK-8 injection, evidence that CCK-8 exerts its effects by directly activating cholecystokinin (CCK) receptors localized on anterior pituitary corticotrophs rather than in brain or the vagus nerve. Subsequent experiments demonstrated further that type A CCK receptors primarily mediate the stimulatory effect of CCK-8 on corticotroph secretion. Thus, devazepide, a selective CCK-A receptor antagonist, produced a dose-related inhibition of the CCK-8-stimulated rise in circulating beta-endorphin concentrations. Less selective CCK-A antagonists, including proglumide and lorglumide, produced little or no effect, however. Unexpectedly, the CCK-B receptor antagonist, L-365,260, enhanced the response to CCK-8, an effect diametrically opposite to that produced by CCK-A antagonists. These observations indicate that CCK-A and CCK-B receptors mediate quite different, if not opposing, roles in regulating corticotroph secretion.

The related rat cholecystokinin (CCK)-A and gastrin/CCK-B receptors can be selectively blocked by the antagonists L364718 and L365260, respectively. In order to determine receptor domains which are important in conferring specificity for L365260 and L364718 we constructed by overlap-PCR a rat gastrin/CCK-B receptor chimaera which contained the seventh transmembrane domain of the rat CCK-A receptor. Receptor binding assays on transiently transfected COS cells demonstrated a selective reduction in the affinity of the chimaeric receptor for L364718, so that the L365260 and L364718 affinities were of a similar order. Since the chimaera differs from the wild-type gastrin/CCK-B receptor by only six amino acids we conclude that one or more of these six amino acids contributes to L364718 binding and that the affinity determinants of L365260 and L364718 must, at least in part, be different. Furthermore, the affinity of the chimaera for gastrin is essentially the same as the gastrin/CCK-B receptor, indicating that the six different amino acids probably do not contribute to peptide agonist binding.

Gallbladder contraction and hormone release were measured in six healthy volunteers after their ingestion of two commercially available fatty meals (Biloptin Fatty Meal and Sorbitract) and intravenous bolus injection of 1 Ivy Dog Unit/kg body weight cholecystokinin (CCK) to compare the effectiveness of fatty meals to CCK. Differences in gallbladder volumes, rate constants of emptying, and times of maximal contractions, as measured by real-time sonography, were not statistically significant. Peak levels of CCK, a potent stimulant of gallbladder contraction, and of pancreatic polypeptide, an inhibitor of gallbladder contraction, were significantly higher (P less than .05-P less than .005) after administration of CCK than after ingestion of each fatty meal, but this did not significantly affect emptying rate or maximal contraction. We conclude that the use of intravenous CCK does not offer any advantage over the ingestion of fatty meals in radiographic studies of gallbladder involving induced contractions.

In this paper report the effects of peripheral (intraperitoneal, i.p.) and central (intracerebroventricular, i.c.v.) injection of selective cholecystokinin (CCK) receptor agonists on food intake in the rat. Stimulation of peripheral and central CCK-A receptors by the selective CCK-A receptor agonist A-71623 suppressed intakes of a liquid diet in both deprived and sated rats. In contrast, i.c.v., but not i.p., injections of the selective CCK-B receptor agonist A-63387, reduced food intakes, although on a molar basis the effect was much less than that seen with A-71623. Although these results stress the relative importance of the CCK-A receptor in the effects of exogenous CCK-8 administration on feeding, stimulation of the CCK-B receptor may still be involved in the control of feeding following the endogenous release of CCK.

The effects of cholecystokinin (CCK) agonists and antagonists on spontaneous and electrically evoked endogenous GABA release from rat cerebral cortex slices were evaluated. Neither the nonselective and CCK(B)-selective receptor agonists CCK-8S (3-1,000 nM) and CCK-4 (3-1,000 nM), respectively, nor the selective CCK(B) and CCK(A) receptor antagonists GV 150013 (3-30 nM) and L-364,718 (10-100 nM), respectively, significantly affected spontaneous GABA release. CCK-8S (1-1,000 nM) and CCK-4 (1-1,000 nM) increased the electrically (5 and 10 Hz)-evoked GABA release. On the contrary, GV 150013 (10 and 30 nM) significantly decreased the electrically evoked GABA release only when the slices were stimulated at the higher 10 Hz frequency. The CCK-8S- and CCK-4-induced increases in electrically evoked GABA release were counteracted by GV 150013, but not by L-364,718. Furthermore, GV 150013 at 3 nM shifted to the right the CCK-4 concentration-response curve, whereas at the higher 10 nM concentration it dramatically flattened the curve. Finally, in cortical slices obtained from rats chronically treated with GV 150013, the concentration-response curve of CCK-4 was shifted to the left and the peak effect of the peptide was significantly higher than that observed in naive animals. These results suggest that CCK increases electrically evoked, but not spontaneous, endogenous GABA release from rat cortical slices, possibly by activating local CCK(B) receptors. In addition, chronic treatment with the novel CCK(B) receptor antagonist GV 150013 leads to an enhanced responsiveness of cortical slices to CCK-4 application.

Otsuka Long-Evans Tokushima Fatty (OLETF) rat lacking CCK-A receptors are hyperphagic and obese. Previous work has demonstrated alterations in neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA expression in ad libitum fed OLETF rats compared to lean Long-Evans Tokushima Otsuka (LETO) controls. In order to determine whether alterations in sensitivity to central peptides involved in overall feeding control may contribute to the hyperphagia and obesity in OLETF rats, we assessed OLETF and LETO rats feeding responses to lateral ventricular infusions of NPY (1 and 3.2 nmol), the melanocortin 3/4 agonist MTII (0.1 and 0.32 nmol) and the melanocortin receptor antagonist SHU-9119 (0.25 and 0.5 nmol). At a 3-h time point, NPY increased food intake in both OLETF and LETO rats. OLETF rats were more sensitive, having significant increases at both NPY doses and a greater increase at the higher dose. The melanocortin agonist MTII decreased intake in both LETO and OLETF rats. At the 20-h time point, the magnitude of suppression was greater in OLETF rats. SHU-9119 increased food intake in both groups. OLETF rats were more sensitive with larger relative increase and longer-lasting effects at the lower dose. These results are consistent with demonstrated alterations in neuropeptide gene expression in OLETF rats and indicate that alterations in responsivity to NPY and melanocortin signaling are unlikely to contribute to their hyperphagia and obesity.

The selective type A and B cholecystokinin (CCK) receptor antagonists L364,718 and L365,260 were used to identify the receptor subtype that mediates the satiety effect of endogenous CCK. Male rats (n = 12-13/group), fed ground rat chow ad lib, received L364,718 (0, 1, 10, 100, or 1000 micrograms/kg IP) or L365,260 (0, 0.1, 1, 10, 100, 1000, or 10,000 micrograms/kg IP) 2 h after lights off, and food intake was measured 1.5, 3.5, and 5.5 h later. L364,718 significantly stimulated 1.5-h food intake by more than 40% at 10 micrograms/kg and higher doses; cumulative intake at 3.5 and 5.5 h remained elevated by about 20% at 1000 and 100 micrograms/kg of L364,718, respectively. In contrast, L365,260 had no significant stimulatory effect on feeding at any dose. The potency of L365,260 for antagonizing gastrin-stimulated gastric acid secretion was examined in unanesthetized rats. Male rats (n = 14), prepared with gastric and jugular vein cannulas, received doubling doses of gastrin (G-171) (0.16-5 nmol/kg/h IV), each dose for 30 min, and gastric juice was collected for each 30-min period. G-171 stimulated gastric acid output dose dependently; the minimal effective dose was 0.16 nmol/kg/h, while maximal output (5-fold above basal) occurred at 5 nmol/kg/h. L365,260 (0, 1, 10, 100, 1000, or 10,000 micrograms/kg IV), administered 30 min before continuous infusion of G-171 (1.25 or 5 nmol/kg/h), significantly inhibited acid output only at 10,000 micrograms/kg; cumulative 60-min output was decreased by 60%. These results suggest that CCK acts at CCK-A receptors to produce satiety during the dark period in ad lib-feeding rats.

The involvement of the cholecystokinin (CCK)-A receptor in fever was studied. The polyphasic febrile responses to lipopolysaccharide (LPS; 10 microg kg-1, I.V.) were compared between wild-type Long-Evans (LE) rats and the CCK-A-receptor-deficient Otsuka LE Tokushima Fatty (OLETF) rats. The response of the wild-type rats was biphasic, which is typical for LE rats. Phases 1 and 2 of the response of the OLETF rats were similar to those of the LE rats, but the OLETF rats also developed a robust phase 3. This late enhancement of the febrile response could reflect either the absence of the A receptor per se or a secondary trait of the mutant strain. To distinguish between these possibilities, we conducted a pharmacological analysis. We studied whether the normally low phase 3 of LE rats can be enhanced by a CCK-A-receptor antagonist, sodium lorglumide (4.3 microg kg-1 min-1, 120 min, I.V.), and whether the normally high phase 3 of Wistar rats can be attenuated by a CCK-A receptor agonist, sulphated CCK-8 (up to 0.17 microg kg-1 min-1, 120 min, I.V.). The dose of sodium lorglumide used was sufficient to increase food intake (to block satiety), but it did not affect the fever response. In both febrile and afebrile rats, CCK-8 induced dose-dependent skin vasodilatation and decreased body temperature, but it failed to produce any effects specific for phase 3. We conclude that the exaggeration of phase 3 in OLETF rats reflects a secondary trait of this strain and not the lack of the CCK-A receptor per se. None of the three known phases of the febrile response of rats to LPS requires the CCK-A receptor.

Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

1. The effect of amphetamine on gastrointestinal (GI) transit and the plasma levels of cholecystokinin (CCK) were studied in male rats. 2. Gastric emptying was inhibited both acutely and chronically by the administration of amphetamine. GI transit was decreased by the acute administration of amphetamine but not affected by the chronic administration of amphetamine. 3. Plasma CCK levels were increased dose-dependently by amphetamine. 4. Proglumide, a CCK receptor antagonist, prevented amphetamine-induced inhibition of gastric emptying and the decrease in GI transit in male rats. 5. The selective CCK(A) receptor antagonist, lorglumide, dose-dependently attenuated the amphetamine-induced inhibition of gastric emptying in male rats. In contrast, the selective CCK(B) receptor antagonist, PD 135,158, did not reverse the effect of amphetamine on gastric emptying. 6. Both lorglumide and PD 135,158 reversed the inhibitory effect of amphetamine on GI transit in male rats. 7. These results suggest that amphetamine-induced inhibition of gastric emptying and intestinal transit is due in part to a mechanism associated with the hypersecretion of endogenous CCK.

Previously we demonstrated that glutamatergic and noradrenergic receptors mediate the relay of visceral information through the parabrachial nucleus (PBN) and that calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), neurotensin (NT), and cholecystokinin (CCK) may modulate these responses. The interactions of these neurotransmitters and neuropeptides were examined in male Wistar rats (17) that were anesthetized with chloral hydrate and ventilated and in which blood pressure and heart rate were continuously monitored. The left cervical vagus nerve was stimulated at submaximal current intensities to elicit changes in single and multiunit activity of visceral thalamic neurons (VTNs). Peristimulus-time and continuous-time histograms of VTN activity were made before and after 200-nl injections of peptides, neurotransmitter agonists or antagonists, or artificial cerebrospinal fluid into the PBN. Combined injection of CGRP and SP into the PBN produced a synergistic inhibition of spontaneous VTN activity and the vagally evoked VTN response. Combined injection of NT and phenylephrine (PE) into the PBN produced only an additive increase in the spontaneous activity of VTNs. Prior administration of SOM in the PBN blocked the excitatory action of an alpha-adrenergic agonist (phenylephrine) injection on the spontaneous activity of VTNs, whereas CGRP, SP, or CCK had no effect on the alpha-agonist-induced response. Prior injection of an alpha-adrenergic antagonist (phentolamine) prevented the excitatory effect of NT in the PBN. Injection of CGRP, SP, NT, or CCK into the PBN did not change the response of VTNs to application of glutamate. These results suggest mechanisms for peptide interaction with primary neurotransmitters in the PBN and indicate whether the neuropeptides are acting before the primary neurotransmitter synapse or postsynaptically.

Boc-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2-phenylether ester (CCK-JMV-180) has been reported to be a CCK-based heptipeptide with novel in vitro properties. Based on studies conducted in rat and mouse pancreatic acini, it has been proposed that the compound acts as an agonist at the high-affinity site and an antagonist at the low-affinity site in the rat, but as an agonist at both sites in the mouse. In the present study, we examined the effects of CCK-JMV-180 on locomotor activity in the rat and on intake of a liquid diet in the rat and mouse. Although CCK-JMV-180 slightly reduced activity on its own in the rat, it completely reversed the suppression produced by coadministration of CCK-8. In rat feeding studies, CCK-JMV-180 failed to suppress intakes of a liquid diet, but was able to antagonize the anorectic effects of CCK-8. In contrast, in the mouse CCK-JMV-180 potently suppressed intakes on its own, and this effect was blocked by pretreatment with the selective CCK-A receptor antagonist, A-70104. The results of these studies suggest that similar receptor mechanisms are involved in CCK's ability to inhibit food intake in vivo and its effects on pancreatic function in vitro.

Previously, we have showed that the cholecystokinin (CCK)-A receptor expression in hypothalamus is closely related with the responsiveness of electroacupuncture (EA)-mediated analgesic effects in rats. In order to confirm this observation more directly in vivo, the EA-mediated analgesic effects are compared between Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the natural knockout rats with the homozygously disrupted CCK-A receptor gene, with Long-Evans Tokushima Otsuka (LETO) rats. They were stimulated at the zusanli (ST36) acupoint without using anesthetics or holders. The tail flick latency (TFL) test was performed to quantify analgesic effects and then the mean TFL increase ratios were calculated. OLETF rats showed a mean increase of 53% and LETO rats showed a mean increase of 31% of TFL. Our results suggest that the analgesic effect of acupuncture is closely related with the amount of CCK-A receptor expression.