Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8>> bombesin>> carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol>> bombesin>> CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.

OBJECTIVE

In the general adaptation syndrome, gastric lesions are the first manifestation of stress. We hypothesized that acute pancreatitis (AP), an inflammatory acute disease, will be exacerbated if unchained following stress. Visceral hypersensitivity will be enhanced due to catecholaminergic discharges leading to an over-induction of the intrapancreatic cholinergic tone with increased response of the pancreocyte to cholecystokinin (CCK). Our aim was to investigate the influence of stress before AP on the later AP, and the effect of AP on underlying diseases such as gastric ulceration.

METHODS

The model of stress induced by restraint was followed by the bilio-pancreatic duct outlet exclusion closed duodenal loops model. The effect of autonomous arc reflex (AAR) interruption by anesthetics after stress but before AP was assessed. The participation of the vagal and sympathetic pathways and involvement of CCK-A receptors were considered. The degree of severity was evaluated using biochemical and histopathological analyses.

RESULTS

Induction of AP after stress was more severe than in its absence. Acinar and fat necrosis, hemorrhage and neutrophil infiltrate foci were evenly distributed, being significantly greater in size and number after stress. Gastric ulceration evolved to ulcer, hemorrhage and gastric necrosis after AP triggering. Serum amylase, lipase, C-reactive protein, IL-6, IL-10 and plasmatic hsp72 as well as pancreatic and lung myeloperoxidase were significantly elevated in AP after stress while pancreatic amylase and lipase were significantly reduced. AAR blockage ameliorated AP after stress.

CONCLUSIONS

Stress aggravates pancreatic pathology while AP deteriorates gastric pathology, and anesthetic treatment was beneficial for both. Restraint in other animal models can be useful to study the influence of stress in the evolution of other diseases.

BACKGROUND

Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice.

METHODS

A liquid gastric load prepared with phenol red was administered via an orogastric tube (0.15 ml/mouse). The animals were killed by decapitation, and gastric emptying was estimated at 10 and 30 min after ingestion. The effects of the sulfated form of CCK-8 (CCK-8S) and of graded doses of atropine were examined. In addition, a proton pump inhibitor was administered to wild-type mice to examine the contribution of gastric acid to emptying.

RESULTS

Gastric emptying was significantly enhanced in mice lacking CCK-BR, as compared with wild-type and CCK-AR(-/-) mice. CCK-8S inhibited gastric emptying in mice with CCK-AR, but not in mice without CCK-AR. A proton pump inhibitor did not affect gastric emptying. Atropine dose dependently inhibited gastric emptying in all genotypes. The thickness of smooth muscle was comparable for all genotypes.

CONCLUSIONS

The gastric emptying of a nonnutrient liquid load was enhanced in mice without CCK-BR, although the precise mechanism is not known. Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice.

In this study the muscarinic receptor antagonist atropine and the cholecystokinin (CCK)-A receptor antagonist loxiglumide were used to investigate the relative importance of cholinergic and CCK-mediated regulation of intestinal phase antro-pyloro-duodenal motility. Plasma levels of gastrointestinal hormones [pancreatic polypeptide (PP), gastrin, CCK] were concomitantly determined to estimate biological potency of the doses of the receptor antagonists. In eight healthy male volunteers, a 30-min basal interdigestive period was followed by duodenal perfusion of a mixed liquid meal for 150 min at 1.6 kcal min-1 against a background of saline or atropine (5 micrograms kg-1 h-1) or loxiglumide (10 mg kg-1 h-1). Antropyloro-duodenal motility was continuously monitored with a sleeve straddling the pylorus. Against a background of saline, duodenal nutrients persistently stimulated isolated pyloric pressure waves. After 60 min, the initially low antral and duodenal contraction rates increased. In the fed state, atropine reduced total number of antral contractions and integrated motility index by 73% (P < 0.01) and 76% (P < 0.005), total number of pyloric contractions and integrated motility index by 43% and 50% (P < 0.05) with inhibition increasing over time. It did not alter duodenal contraction frequency but diminished duodenal motility index by 39% (P < 0.05) owing to a reduction in amplitude and duration of contractions. Loxiglumide decreased total numbers of antral, pyloric and duodenal contractions by 44% (P < 0.05), 74% (P < 0.005) and 41% (P < 0.005) respectively. It reduced cumulative antral, pyloric and duodenal motility indexes by 60% (P < 0.01), 80% (P < 0.01) and 61% (P < 0.05) respectively. Atropine fully abolished PP release to duodenal nutrients whereas loxiglumide reduced it by 60% (P < 0.05). Both atropine and loxiglumide enhanced gastrin release whereas only loxiglumide markedly stimulated CCK release. We conclude that both cholinergic input and endogenous CCK are major stimulatory regulators of antro-pyloroduodenal motility in the intestinal phase. There appears to be a regional heterogeneity of cholinergic and CCK control. Cholinergic input predominates in the antrum. Both systems are equipotent at the pylorus. CCK predominates in the duodenum. We suggest that CCK primarily interacts with receptors on cholinergic neurons in the antropyloric region and primarily affects smooth muscle receptors in the duodenum.

BACKGROUND

It has been assumed that gastrin stimulates the growth of pancreatic cancer in an autocrine way through co-expression of gastrin and the cholecystokinin-B receptor (CCK-BR). However, pancreatic cancer cell lines established directly from patients have revealed a great heterogeneity in cell proliferation when exposed to CCK, gastrin and their receptor antagonists. The aim of this study was therefore to examine co-expression of CCK-A and CCK-B receptor (CCK-AR and CCK-BR), and gastrin mRNA as well as the secretion of CCK and gastrin peptides in these cell lines.

METHODS

Fourteen cell lines were established from primary pancreatic cancers or their metastases. Total RNA was isolated from the cell lines and reverse-transcribed into single-stranded cDNA. A PCR technique based on Taq polymerase-antibody interaction and CCK-AR, CCK-BR and gastrin-specific primers, followed by Southern blot analysis, were the methods used. The incubation mediums were analysed for the presence of secreted CCK/proCCK and gastrin/progastrin peptides by specific radioimmunoassays (RIA).

RESULTS

By means of nested Reverse-Transcribed Polymerase Chain Reaction (nested RT-PCR), combined with Southem blot analysis of the PCR amplified products, CCK-AR and gastrin mRNA co-expression was detected in cell lines LPC-6p and LPC-10m, whereas CCK-BR and gastrin mRNA could be detected in cell lines LPC-8p and LPC-12m. A low level of secreted CCK peptides was detected in cell line LPC-6p, which also expressed CCK-AR mRNA. In no other cases were CCK or gastrin peptides detected in the cell culture mediums.

CONCLUSIONS

The lack of CCK-BR and gastrin mRNA co-expression, and not detectable levels of secreted CCK and gastrin in culture media, does not lend support to the hypothesis that concomitant gene-expression of CCK receptors and gastrin or CCK are essential to maintaining pancreatic cancer cell proliferation.

Cholecystokinin (CCK) is a gut peptide hormone known to stimulate postprandial gallbladder contraction and pancreatic enzyme secretion. It has also been shown to induce the growth of normal pancreas and of malignant and premalignant lesions in rodents. Although CCK has been shown to promote the growth of human adenocarcinoma cell lines, its role in the growth of human pancreatic adenocarcinomas in vivo is less clear. Localization of CCK receptors to neoplastic cells within resected human tissue specimens would be suggestive of its potential action as an in vivo promoter of human pancreatic cancer. Resected tissue specimens of pancreatic adenocarcinomas were therefore studied by both reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization for the presence of CCK-A receptors. Ninety percent of studied tumors demonstrated CCK-A expression by RT-PCR, and this expression was localized to neoplastic cells by in situ hybridization. An increase in the expression of CCK receptors is a mechanism by which pancreatic malignancies may gain a significant growth stimulus.

Whole-cell patch-clamp recordings were performed to study ionic and molecular mechanisms by which cholecystokinin (CCK) peptides modulate the membrane excitability of acutely dissociated rat neostriatal neurons. Immunohistochemical staining studies indicated that about 95% of acutely isolated neostriatal neurons were GABA(gamma-aminobutyric acid)ergic medium-sized cells. During current-clamp recordings, sulfated cholecystokinin octapeptide (CCK-8) depolarized neostriatal neurons and evoked action potentials. During voltage-clamp recordings, CCK-8 induced inward currents at negative membrane potentials by increasing the voltage-insensitive and non-selective cationic conductance. Cholecystokinin tetrapeptide (CCK-4), a selective CCKB receptor agonist, also evoked cationic currents. The CCK-8-induced cation currents were antagonized by PD135,158 (4-{[2-[[3-(1H-indol-3yl)-2-mehtyl-1-oxo-2-[[[1.7.7.-trimeth yl-bicyclo [2.2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl]amino-4- oxo- [1S-1 alpha, 2 beta [S*(S*)]4 alpha]}-butanoate N-methyl-D-glucamine), a highly specific and potent CCKB receptor antagonist. The CCK-8-evoked inward currents were blocked by the internal perfusion of 1 mM GDP-beta-S. In neostriatal neurons dialyzed with 0.5 mM GTP-gamma-S, the cationic currents produced by CCK-8 became irreversible. Pretreating neostriatal neurons with 500 ng/ml pertussis toxin did not prevent CCK-8 from evoking cationic currents. Internal administration of heparin (2 mg/ml), an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, and buffering of intracellular calcium with the Ca(2+)-chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 10 mM), suppressed CCK-8-evoked cationic currents. These findings suggest that, by activating CCKB receptors, CCK-8 excites rat neostriatal neurons through enhancing a non-selective cationic conductance and that pertussis toxin-insensitive G-proteins mediate CCK-8 enhancement of the cationic conductance. The coupling mechanism via G-proteins is likely to involve the production of IP3, and the subsequent IP3-evoked Ca2+ release leads to the opening of non-selective cation channels.

The mechanism by which acid in the duodenum inhibits proximal gastric motor function and delays emptying was investigated in urethan-anesthetized and awake rats. Gastric motility inhibited by duodenal acid (0.2 N HCl) in urethan-anesthetized rats was attenuated by 68 and 54%, respectively, by functional ablation of the vagal or spinal sensory innervation with capsaicin. 5-Hydroxytryptamine3 receptor blockade with zacopride (0.2 mg/kg ip) or cholecystokinin (CCK)-A-type receptor blockade with MK-329 (1 mg/kg ip) had no effect on the motility response to acid. In awake rats with chronically implanted gastric and duodenal cannulas, perfusion of the duodenum with acid (0.1 and 0.2 N HCl) inhibited gastric emptying of a nonnutrient liquid (38 and 59%, respectively). Blockade of CCK-A-type receptors reduced by 30% inhibition of gastric emptying induced by 0.1 N HCl. However, functional ablation of the vagal or spinal sensory innervation, 5-hydroxytryptamine3 receptor blockade, or immunoneutralization of secretin by systemic administration of a polyclonal antibody (no. 7842, 1 ml ip) had no effect on acid-induced (0.1 N HCl) inhibition of gastric emptying. Perfusion of the duodenum with 0.2 N HCl but not 0.1 N HCl inhibited proximal gastric motility in awake rats. These results suggest that 1) a duodenal acid load inhibits gastric emptying in part by a mechanism involving CCK and 2) decreased proximal gastric motility plays a minor role in inhibition of gastric emptying in response to acid.

A region between residues 38 and 42 of the human cholecystokinin-A (CCK-A) receptor was shown to be involved in the binding of CCK but not in that of JMV 179 and JMV 180, two peptides closely related to CCK (Kennedy, K., Escrieut, C., Dufresne, M., Clerc, P., Vaysse, N., and Fourmy, D. (1995) Biochem. Biophys. Res. Commun. 213, 845-852). In the present study, we have identified the residues of both the receptor and the ligand responsible for this differential binding. Residues Trp-39 and Gln-40 of the receptor were crucial for binding of the C-terminal nonapeptide of CCK as W39F and Q40N mutants demonstrated parallel decreases in both affinity and potency to induce accumulation of inositol phosphates (12.9- and 20.9-fold). The W39F and Q40N mutant receptors bound CCK analogues modified at their C-terminal end, including JMV 179 and JMV 180, as well as the C-terminal amidated heptapeptide of CCK, with identical affinities to the wild-type receptor. In contrast, W39F and Q40N mutants bound CCK octapeptide with the same decreased affinity as the CCK nonapeptide. The modeling of the CCK-A receptor and the docking of the peptide agonists [Thr,Nle]CCKCCK-8 indicated that their N terminus was connected to the receptor through a strong bond network involving Trp-39 and Gln-40 thus confirming experimental data. These first molecular data identifying the agonist binding site of the human CCK-A receptor represent an important step toward the complete delineation of the agonist binding site and the understanding of the molecular mechanisms that govern differential activation of this receptor by CCK-related peptides.

The contractile response of the isolated guinea-pig ileum to cholecystokinin octapeptide (CCK-8) that remained in the presence of atropine was greatly inhibited by the substance P antagonist D-Pro2,D-Trp7,9-substance P. This indicates that the atropine-resistant contraction to CCK-8 is mediated by the release of substance P from enteric neurones. Activation of alpha-adrenergic receptors by noradrenaline, clonidine or mesenteric sympathetic nerve stimulation inhibited the atropine-resistant contraction to CCK-8 but did not affect the contractile effect of substance P. It is concluded that alpha-adrenergic receptors exert an inhibitory influence on the release of substance P from enteric neurones.

The activity of SR 27897, a potent and selective CCK-A vs CCK-B receptor antagonist (Ki = 0.2 nM on guinea-pig pancreas vs 2000 nM on rat brain) was studied on behavioural, electrophysiological and biochemical effects induced by peripheral or central injection of CCK-8S. For comparative purposes, devazepide, a reference CCK-A receptor antagonist, was investigated in these same models. CCK-induced hypophagia and CCK-induced hypolocomotion in rats, two behavioural changes associated with the stimulation of peripheral CCK-A receptors, were dose-dependently antagonized by SR 27897 (ED50 = 0.003 and 0.002 mg/kg i.p., respectively) and devazepide (ED50 = 0.02 and 0.1 mg/kg i.p., respectively). CCK-induced decrease of cerebellar cGMP levels in mice was also reduced by SR 27897 (ED50 = 0.013 mg/kg) and by devazepide (0.084 mg/kg). The CCK-induced turning behaviour after intrastriatal injection in mice, and the potentiation of the rate suppressant activity of apomorphine on rat DA neurons, were blocked by higher doses of SR 27897 and devazepide, consistent with the probable central origin of these effects. The respective ED50s were 0.2 mg/kg i.p. for SR 27897 and 4.9 mg/kg i.p. for devazepide in the former model, while the respective minimal effective doses were 1.25 and 5 mg/kg i.p. in the latter test. In most tests the i.p./p.o. ratio for SR 27897 was near unity, suggesting a high oral bioavailability of the compound. Taken together, these findings support the notion that SR 27897 behaves as a potent CCK-A antagonist able to cross the blood brain barrier.

To investigate whether activated autologous platelet-rich plasma (PRP) can promote proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in vitro. hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3(rd) passage. PRP was collected and activated from human peripheral blood of the same patient. Cultured hASCs were treated with normal osteogenic inductive media alone (group A, control) or osteogenic inductive media plus 5%, 10%, 20%, 40%PRP (group B, C, D, E, respectively). Cell proliferation was assessed by CCK-8 assay. mRNA expression of osteogenic marker genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) and core binding factor alpha 1 (Cbfa1) were determined by Real-Time Quantitative PCR Analysis (qPCR). Data revealed that different concentrations of activated autologous PRP significantly promoted hASCs growth in the proliferation phase compared to the without PRP group and resulted in a dose-response relationship. At 7-d and 14-d time point of the osteogenic induced stage, ALP activity in PRP groups gradually increased with the increasing of concentrations of PRP and showed that dose-response relationship. At 21-d time point of the osteogenic induced stage, PRP groups make much more mineralization and mRNA relative expression of ALP, OPN, OCN and Cbfa1 than that without PRP groups and show that dose-response relationship. This study indicated that different concentrations of activated autologous PRP can promote cell proliferation at earlier stage and promote osteogenic differentiation at later stage of hASCs in vitro. Moreover, it displayed a dose-dependent effect of activated autologous PRP on cell proliferation and osteogenic differentiation of hASCs in vitro.

Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.

The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.

In the present article, we have synthesized three different series of pyrazolo[3,4-b]pyridines and their structural analogues using novel synthetic strategy involving one-pot condensation of 5,6-dihydro-4H-pyran-3-carbaldehyde/2-formyl-3,4,6-tri-O-methyl-D-glucal/chromone-3-carbaldehyde with heteroaromatic amines. All synthesized compounds were evaluated for their anti-inflammatory activity against TNF-alpha and IL-6. Out of 28 compounds screened, 40, 51, 52 and 56 exhibited promising activity against IL-6 with 60-65% inhibition at 10 microM concentration. Amongst these, 51, 52 and 56 showed potent IL-6 inhibitory activity with IC(50)'s of 0.2, 0.3 and 0.16 microM, respectively. Compound 56 was not cytotoxic in CCK-8 cells up to the concentration of >100 microM.

The present study was undertaken to investigate how the activation of gastric mechanoreceptors by distension of the stomach in conscious gastric fistula rats influences gastric emptying; and the roles of capsaicin sensitive vagal afferent fibres and the 5-HT3, GRP and CCK-A receptors involved in mediating these responses. To activate mechanoreceptors by non-nutrient dependent pathways, methylcellulose in saline was used to distend the stomach (5 cm H2O) and the subsequent emptying of saline was examined immediately, and at 3, 5 and 10 min following distension. Prior distension delayed the subsequent emptying of saline instilled into the stomach compared with non-distended controls (2.28+/-0.09 ml/5 min; P < 0.001). Topical application of capsaicin, completely abolished the distension-induced inhibition of gastric emptying when compared with vehicle treated rats (2.82+/-0.09 vs. 2.38+/-0.04 ml/5 min; P < 0.001). Peripheral administration of a GRP antagonist (2258 U89UJ, 1 mg/kg), and a 5-HT3 antagonist (BRL4369UA, 50 microg/kg) significantly reversed (2.56+/-0.14 ml/5 min; P < 0.05 and 2.61+/-0.07 ml/5 min; P < 0.01; respectively) the delay in gastric emptying induced by distension. When the rats were treated with the CCK-A antagonist, gastric emptying of saline following distension was also significantly facilitated (2.56+/-0.07 ml/5 min; P < 0.001). In contrast, the CCK-B/gastrin receptor antagonist had no significant effect on the distension induced delay in gastric emptying (1.95+/-0.12 ml/5 min). The present results suggest that gastric distension in conscious gastric fistula rats delays gastric emptying by activating capsaicin-sensitive extrinsic afferent nerve fibres. Moreover, the results also indicate that distension-induced mechanisms involve GRP, 5-HT3 and CCK-A receptors, but not CCK-B receptors.

We have previously reported that CCK-A receptor agonists and CCK-B receptor antagonists both enhance memory in an olfactory recognition test. Here, we report that the memory-enhancing effect of the CCK-B receptor antagonist L-365,260 (1 mg/kg i.p.), but not that of the CCK-A receptor agonist caerulein (0.03 mg/kg i.p.), was dramatically decreased following a bilateral transection of the perforant path, a principal source of input to the hippocampal formation. We further confirmed that a significant memory deficit occurred subsequent to this deafferentation of the hippocampus in untreated animals. In contrast, the effect of caerulein, but not that of L-365,260, was abolished following a bilateral subdiaphragmatic vagotomy. These results demonstrate that the hippocampal system plays a role in olfactory recognition and indicate that distinct neuronal pathways underlie the memory-enhancing effects of CCK-A and CCK-B drugs observed in the olfactory recognition test. The former effects (CCK-A) appear to involve a peripheral relay to the brain via the vagus nerve, whereas the latter (CCK-B) are directly central and involve, at least in part, the hippocampal system.

OBJECTIVE

To study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalphaalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed.

METHODS

pECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalphaalphaalpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalphaalphaalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader.

RESULTS

Cells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaB<em>alpha</em>, CFP-IkappaB<em>alpha</em>M, and CFP-IkappaB<em>alpha</em>243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaB<em>alpha</em>244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-<em>alpha</em>, p53 and Bax in CFP-IkappaB<em>alpha</em> transfected cells did not change significantly, while in YFP-p65/CFP-IkappaB<em>alpha</em> co-transfected cells, IkappaB<em>alpha</em> decreased the transcription of p65 downstream gene TNF-<em>alpha</em> (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaB<em>alpha</em> (IkappaB<em>alpha</em>M) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaB<em>alpha</em> (IkappaB<em>alpha</em>243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-<em>alpha</em> (1.51+/-0.203) a little bit. However, the C terminus of IkappaB<em>alpha</em> (IkappaB<em>alpha</em>244C) increased the transcription of NF-kappaB, TNF-<em>alpha</em>, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The <em>CCK</em>-8 experiment also showed that IkappaB<em>alpha</em>244C and p53 synergistically mediate apoptosis.

CONCLUSIONS

IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalphaalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalphaalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Trichosanthes kirilowii tuber is one of most popular herbal plant of East Asia, which has been prescribed for patients with diabetes, rigorous coughing, breast abscesses, and cancer-related symptoms.

OBJECTIVE

To investigated the anticancer properties of the methanol extract of Trichosanthes kirilowii tuber (TKE), focusing on cell cycle arrest and microtubule instability in HepG2 cells.

METHODS

Cell growth and death were checked using a CCK-8 assay and a LDH release assay respectively. Cell cycle was analyzed by FACS after PI staining. Immunofluorescence, Western blot, real-time PCR for tubulin were performed.

RESULTS

TKE treatment inhibited cell growth at around 25 microg/mL of IC50 in a CCK-8 assay and a LDH release assay, but did not result in cell death. We found that TKE induced cell cycle arrest at the G2/M phase in a time-dependent manner. However, an immunofluorescence assessment of beta-tubulin revealed a dramatically reduced amount of polymerized tubulin after TKE treatment. Furthermore, TKE treatment radically decreased the polymerized portion of soluble tubulin in a dose-dependent manner, as did colchicine; the effects, however, were opposite to those of paclitaxel in comparative analysis of polymerized to soluble tubulin. We also found that TKE treatment moderately affected alpha-tubulin protein production, but not that of beta-tubulin and its gene expression using a Western assay and real-time PCR.

CONCLUSIONS

Anticancer mechanisms of TKE linked to the inhibition of tubulin polymerization, through which it exerts cell cycle arrest at the G2/M phase in the HepG2 cell line.

Estrogen-related receptor alpha (ERRα) plays an important role in the development of hormone-dependent cancers, but its roles in lung cancer remain elusive. The present study was aimed to investigate the effects of ERRα on the proliferation and metastasis of lung cancer A549 cells. The mRNA and protein levels of ERRα were detected in lung cancer A549 and MCF-7 cells and bronchial epithelial BEAS-2B cells by qRT-PCR and Western blotting, respectively. ERRα plasmid transfection and XCT-790 (an inverse agonist of ERRα) were used to up-regulate or down-regulate ERRα expression in A549 cells, respectively. The viability of A549 cells was measured by cell counting kit-8 (CCK-8) and the motility of A549 cells by wound healing assay and Transwell migration/invasion assay. The epithelial markers E-cadherin (E-Cad) and zona occludin-1 (ZO-1), the mesenchymal markers fibronectin (FN) and vimentin (Vim) and the transcription factors (Snail, Zeb1 Twist and Slug) were further detected at mRNA and protein levels by qRT-PCR and Western blotting, respectively. The results showed that ERRα promoted the growth of lung cancer A549 cells in vitro. XCT-790 significantly inhibited the migration and invasion of A549 cells. Over-expression of ERRα promoted the epithelial-to-mesenchymal transition (EMT) of A549 cells, down-regulated the epithelial makers E-Cad and ZO-1, and up-regulated the mesenchymal makers FN and Vim. Silencing of Slug, but not other transcription factors, significantly abolished the ERRα-induced EMT of A549 cells. It was suggested that ERRα promoted the migration and invasion of A549 cells by inducing EMT, and Slug was involved in the process. Targeting ERRα might be an efficient approach for lung cancer treatment.

Menetrier's disease is characterized by giant gastric folds with foveolar hyperplasia and cystic dilatation, hypoproteinemia, and enhanced mucus secretion. The etiology remains unresolved and an effective treatment has yet to be established. Here we show that histamine H(2)-receptor deficient mice developed gastric pathophysiological changes resembling Menetrier's disease for up to 17 months of observation. Mutant mice were found to have an increased stomach weight, enlarged gastric folds with cystic dilatation, hypergastrinemia, hypoalbuminemia, increased mucus secretion and overexpression of mucosal transforming growth factor (TGF) alpha. Both a cholecystokinin (CCK)(2)-receptor antagonist and an epidermal growth factor (EGF)-receptor tyrosine kinase inhibitor significantly reduced the increase in stomach weight. It appears that lack or downregulation of histamine H(2)-receptors might be involved in the pathogenesis of Menetrier's disease.

Pyruvate kinase M2 (PKM2) has been wildly verified to modulate glycolysis in tumor cells. However, the role of PKM2 on the glycolysis of osteoarthritis (OA) chondrocytes is still unclear. In present study, we investigate the function of PKM2 on OA chondrocyte glycolysis and the collagen matrix generation in vitro. Results showed that PKM2 was upregulated in OA chondrocytes compared with healthy control chondrocytes. In OA chondrocytes, ATP expression was lower compared with healthy control chondrocytes. Loss-of-function experiment showed that PKM2 knockdown mediated by lentivirus transfection could significantly suppress the glucose consumption and lactate secretion levels and decrease glucose transporter-1 (Glut-1), lactate dehydrogenase A (LDHA), and hypoxia inducible factor 1-alpha (HIF-1α), indicating the inhibition of PKM2 knockdown on glycolysis. Moreover, Cell Counting Kit-8 (CCK-8), flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay showed that PKM2 knockdown inhibited OA chondrocyte proliferation and promoted the apoptosis. Western blot and immunocytochemical staining showed that PKM2 knockdown downregulated the expression levels of COL2A1 and SOX-9. In summary, our results conclude that PKM2 modulates the glycolysis and extracellular matrix generation, providing the vital role of PKM2 on OA pathogenesis and a novel therapeutic target for OA.

We recently found a specific strain of rats (Otsuka Long Evans Tokushima Fatty; OLETF rats) lacking cholecystokinin (CCK)-A receptor because of a genetic abnormality. Since CCK is an abundant neurotransmitter peptide in the brain, we examined whether these animals exhibit any behavioral abnormalities by conducting open-field tests. OLETF rats showed hypolocomotor activity and a decrease in the incidence of rearing in open-field tests. CCK release from the synaptosomes, and levels of CCK and CCK-B receptor mRNAs were the same as those observed in normal rats. It is suggested that the decreased exploration in OLETF rats may be due to the lack of CCK-A receptor.