Cholecystokinin-8 (CCK-8) dose-dependently increased the cytosolic Ca2+ concentration ([Ca]i) in ventromedial hypothalamic neurons acutely dissociated from the immature rat brain. The CCK-8 response was mimicked by caerulein, but not by CCK(B) agonists, and was often inhibited by CCK(A) receptor antagonists, but rarely by CCK(B) receptor antagonists. The response was dependent on external Ca2+ and Na+, and was inhibited by voltage-dependent Ca2+ channel blockers. The results suggest that CCK-8-induced depolarization via CCK(A) receptors increased Ca2+ influx through a voltage-dependent Ca2+ channel, which in turn increased [Ca]i.

Studies have shown that measurement of thermodynamic parameters (enthalpy, DeltaH degrees and entropy, DeltaS degrees ) can allow discrimination of agonists and antagonists (e.g. Weiland, G.A., Minneman, K.P., Molinoff, P.B., 1979. Fundamental difference between the molecular interactions of agonists and antagonists with the beta-adrenergic receptor. Nature, 281, 114.). Recently, we found that agonists and antagonists were not thermodynamically-distinguished at cholecystokinin (<em>CCK</em>)2-receptors in rat cerebral cortex. However, in this study, the possibility that thermodynamic discrimination at <em>CCK</em>2-receptors exists but that it was not detected, could not be excluded because radioligand binding studies and functional assays were performed in different rat tissues. Therefore, we have repeated these studies using the recombinant <em>CCK</em>2 short isoform (<em>CCK</em>2S)-receptor expressed in NIH3T3 cells, so that ligand affinity (pKI) and intrinsic activity (<em>alpha</em>) measurements could be made in exactly the same receptor system. <em>CCK</em>-8S but not R-L-365,260, S-L-365,260, JB95008, JB93242 or PD134,308 expressed intrinsic activity in an IP assay. The pKD of [3H]-JB93182 decreased with increasing temperature. pKI values for antagonists (R-L-365,260, S-L-365,260, JB95008) and agonists (pentagastrin, <em>CCK</em>-8S) were higher at 4 than at 30 degrees C. There was no effect of temperature on pKI values for the antagonists, PD134,308 and JB93242. Therefore, <em>CCK</em>2-receptor agonists and antagonists at human <em>CCK</em>2S-receptors cannot be discriminated by thermodynamic analysis.

CONCLUSIONS

The results of the present study suggest that the pancreas and parotid glands both respond with hypoplasia during absence of food in the digestive tract and with hyperplasia following pancreatico-biliary diversion (PBD). Factors other than cholecystokinin (CCK) are, however, involved in the effects on the parotid glands, since infusion of CCK-8S and devazepide was without influence.

OBJECTIVE

Total parenteral nutrition (TPN) causes reduced pancreatic weight, whereas PBD evokes hyperCCKemia and enlargement of the rat pancreas. The pancreas and parotid glands have in part similar morphology and function. Therefore, we studied the possible presence of alterations also in the parotid glands during TPN, after PBD and during infusion of sulfated cholecystokinin (CCK-8S) and the CCK-A receptor antagonist devazepide, respectively.

RESULTS

Rats either received TPN for 7 d, or were kept under otherwise identical conditions with free access to food and water. TPN markedly reduced both pancreatic and parotid wet weight and thereby also the protein and amylase contents, and pancreatic DNA content was decreased. There was a significant correlation between the pancreas and parotid glands when comparing these parameters. The concentration of plasma CCK was unaffected by TPN. PBD caused a sevenfold increase in plasma CCK and a three fold increase in the pancreatic weight compared to control rats 28 d after the operation. The protein and DNA contents in the pancreas were found to be increased. The parotid glands increased twofold in weight, but their protein and amylase contents were not affected. There was a significant correlation between the pancreas and parotid glands when comparing weight, and protein and amylase concentrations. Infusion of CCK-8S during 28 d caused a marked increase in pancreatic wet wt and protein and DNA contents. The CCK-A receptor antagonist devazepide induced a reduction in protein and DNA contents in the pancreas. The parotid glands were not affected by either treatment. No effect on the labeling index of serous and ductal cells of the parotid gland was seen at 36 h, 3, 7, and 28 d of infusion with CCK-8S or devazepide.

We describe here the properties of tert-butyloxycarbonyl-Trp-Leu-Asp-Phe-NHNH2 (A-57696), a C-terminal hydrazide analogue of tert-butyloxycarbonyl-CCKAsp-Phe-NH2), at four cholecystokinin (CCK) receptor-bearing tissues, the guinea pig pancreas and gall bladder (Type A), guinea pig cortex (Type B), and NCI-H345 cells, a human small cell lung cancer cell line that expresses CCK-B/gastrin receptors. Using 125I-Bolton-Hunter-cholecystokinin octapeptide (26-33) (125I-Bolton-Hunter-CCKA-57696 was found to be selective for cortical CCK-B receptors (IC50 = 25 nM), compared with pancreatic CCK-A receptors (IC50 = 15 microM). A-57696 behaved as a competitive antagonist in reversing CCKA-57696 (100 microM) did not elicit gall bladder contraction, and it inhibited contractions induced by CCKA-57696 at gall bladder CCK-A receptors was 19 microM. In contrast, A-57696 behaved as a partial agonist (80% of maximal CCKCCK-B/gastrin receptors on NCI-H345 cells. A-57696 and CCKCCKA-57696 were reversed by the CCK-B-selective (R)-L-365,260 (100 nM), whereas at the same concentration, the CCK-A-selective (S)-L-365,260 was ineffective. Binding studies using 125I-Bolton-Hunter-CCKCCKCCKA-57696, and both enantiomers of L-365,260. A-57696 represents a new class of CCK-A peptide antagonist at guinea pig pancreas a new class of CCK-A peptide antagonist at guinea pig pancreas and gall bladder. Its contrasting functional activities at guinea pig CCK-A and CCK-B/gastrin receptors in a human tumor cell demonstrate that, in addition to the previously described differences in binding specificity for selective agonists and antagonists, CCK-A receptors and CCK-B/gastrin receptors have different requirements for activation.

1. The cardiovascular actions of cholecystokinin and related peptides were investigated in the pithed rat. The receptors and the mechanisms involved in these experiments were characterized. 2. Sulphated cholecystokinin octapeptide (sCCK-8, 0.1-100 nmol kg-1, i.v.) elicited a dose-dependent bradycardia and increase in mean arterial blood pressure. Neither gastrin-17 nor pentagastrin had any effect at concentrations up to 100 nmol kg-1. 3. Both the pressor response and bradycardia elicited by sCCK-8 were reduced by the selective CCKA receptor antagonists, devazepide (0.5-50 nmol kg-1) and lorglumide (1-7 mumol kg-1). The selective CCKB receptor antagonists, CI-988 (1 mumol kg-1) and L-365,260 (15 mumol kg-1) did not inhibit the effects of sCCK-8. 4. The pressor response induced with sCCK-8 was reduced by treatment with either phentolamine (3 mumol kg-1) or guanethidine (2 mumol kg-1) and was unaffected by treatment with propranolol, atropine or hexamethonium. The pressor response also persisted following bilateral adrenalectomy. 5. The bradycardia induced with sCCK-8 was unaffected by treatment with phentolamine, propranolol, guanethidine, atropine, hexamethonium or bilateral adrenalectomy. 6. The tetrapeptide of cholecystokinin (CCK-4) elicited a dose-dependent pressor response but did not induce bradycardia. The pressor response was unaffected by devazepide (50 nmol kg-1), L-365260 (15 mumol kg-1) or phentolamine (3 mumol kg-1). 7. In the pithed rat, sCCK-8 acted via CCKA receptors to increase arterial blood pressure indirectly, at least in part, through activation of alpha-adrenoceptors. The observed bradycardia was also mediated byCCKA receptors but possibly through a direct action on the heart.

There is increased interest in the Bn-receptor family because they are frequently over/ectopically expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [D-Tyr(6), β-Ala(11), Phe(13), Nle(14)]Bn(6-14) [Univ.Lig] has the unique property of having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor (CCK(A)R), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCK(A)R or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-π or H-bonding interactions are also important for determining its high affinity.

Porta-caval shunting enhances the trophic effects of cholecystokinin (CCK)-A receptor activation on the pancreas and of CCK-B receptor activation on the ECL cells in the oxyntic mucosa of the rat. The aim of the present study was to study the expression of CCK-A and CCK-B receptor mRNA after porta-caval shunting. Different doses of sulfated CCK-8 (CCK-8s) were administered to porta-caval shunting rats and sham-operated rats, 4 weeks after the operations. The pancreatic wet weight and DNA content were measured and the ECL cells in the oxyntic mucosa were counted after four days of continuous subcutaneous infusion. Total RNA was isolated from pancreas and oxyntic mucosa for Northern blot analysis of CCK-A and CCK-B receptor mRNA. Porta-caval shunting per se did not affect plasma CCK level nor the weight or DNA content of the pancreas, but resulted in increased number of ECL cells despite the fact that the serum gastrin concentration was reduced. The trophic response of the pancreas to low doses of CCK-8s was greater in porta-caval shunted rats than in sham-operated rats. Porta-caval shunted rats displayed an increased CCK-A receptor mRNA concentration in the pancreas (after stimulation with CCK-8s) and an increased CCK-B receptor mRNA concentration in the oxyntic mucosa. In conclusion, the porta-caval shunting-evoked enhancement of the trophic effect of CCK-A receptor activation on the pancreas and of CCK-B receptor activation on the ECL cells is associated with enhanced expression of CCK-A receptor mRNA in the pancreas and of CCK-B receptor mRNA in the oxyntic mucosa.

CCKCCK) in regulating acid output in humans, dose-response curves were constructed to CCKCCK-A receptor antagonist (loxiglumide). During loxiglumide infusion, G17-stimulated acid output was unchanged, whereas CCKCCKCCKCCK-B/gastrin receptor on parietal cells, but at the same time inhibits acid responses by stimulating gastric somatostatin release to a CCK-A receptor-mediated pathway. To test which action of CCK is relevant under physiological circumstances, the effect of loxiglumide on fasting and post-prandial acidity was measured through continuous pH-metry. After eating, gastrin levels increased fourfold compared to controls with concomitant increases in acid secretion. These results suggest that post cibum, CCK is an inhibitor of acid secretion by regulating gastrin through local somatostatin; they support the hypothesis that CCK acts as an enterogastrone.

Our aim was determine the relationship between cholecystokinin (CCK)-A receptor blockade, glucose levels, insulin secretion and gastric emptying in humans, and to assess the effect of CCK-A blockade on pancreatic polypeptide secretion. After a 12-h fast, six healthy volunteers were given [99mTc]iminodiacetic acid monosodium salt (IDA) intravenously (5 mCi). One hour later they were offered a 577 kcal liquid meal containing [99mTc]diethylenetriaminepentaacetic acid (DTPA) (2 mCi) and glucose (105 g). Scintigraphic gastric and gallbladder activity, and plasma glucose, insulin and pancreatic polypeptide responses were monitored. In a second experiment, a continuous intravenous infusion of loxiglumide (7.5 mg kg h(-1)) was started 60 min before and continued until 120 min after test meal ingestion to block the CCK-A receptors. Gallbladder emptying was blocked by loxiglumide. Loxiglumide accelerated gastric emptying, increased insulin secretion without alteration of glucose profiles, and abolished all phases of the postprandial pancreatic polypeptide response. Blockade of peripheral CCK-A receptors accelerates gastric emptying of liquids with an increase in postprandial insulin levels. The lack of changes in glycaemia suggests that alternative homeostatic mechanisms also control postprandial glucose levels. Inhibition of pancreatic polypeptide release may reflect an independent effect of loxiglumide on vagal control involved in pancreatic polypeptide release.

The growth of pancreatic cancers may be influenced by certain gut peptides. However, the alteration of gut peptide receptors in the progress of pancreatic carcinogenesis is largely unknown. With storage phosphor autoradiography, this study visualized and characterized receptors for cholecystokinin (CCK), somatostatin (SST), bombesin (BBS), secretin and vasoactive intestinal peptide (VIP) in pancreata of control hamsters (n = 7) and pancreatic preneoplastic lesions (n = 10) or adenocarcinomas (n = 10) of N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters. The specific CCK-A and secretin receptors expressed in normal pancreata were markedly reduced in pancreatic preneoplastic lesions and absent in adenocarcinomas. In the development of pancreatic tumours, the subgroup of SST receptors did not change, but both the affinity and binding capacity declined. In comparison with the binding of VIP to normal pancreata, specific VIP binding was significantly lower in preneoplastic lesions and almost absent in pancreatic adenocarcinomas. No specific binding for BBS was detected in normal pancreas or (pre)neoplastic lesions of hamster pancreas. The reduction or absence of receptors for CCK, secretin, SST and VIP in hamster pancreas with the progress of carcinogenesis suggests that in BOP-treated hamsters, pancreatic adenocarcinomas have, to a large extent, lost the hormone-dependent characteristics of the original tissue.

Although gallbladder stasis exists in most patients with cholesterol gallstones, it is unknown whether stasis is a causative factor of gallstone disease or merely a consequence of it. We studied the impact of sustained gallbladder stasis induced by a cholecystokinin (CCK)-A receptor antagonist (MK-329) on gallstone formation in ground squirrels fed either a trace or a high-cholesterol diet. MK-329 markedly inhibited gallbladder contraction in vitro in response to CCK (at EC100, control: 3.6 +/- 0.5 vs. MK-329: 1.1 +/- 0.3 g; P < .05) and increased gallbladder fasting volume in vivo (control: 462 +/- 66 vs. MK-329: 1,004 +/- 121 microL; P < .05). Whereas the high-cholesterol diet alone (1%-cholesterol diet + placebo) increased the cholesterol saturation index (CSI) in control animals (trace-cholesterol diet + placebo), MK-329 significantly (P < .05) decreased the CSI in both hepatic and gallbladder bile in animals on the trace-(trace-cholesterol diet + MK-329) as well as on the high-cholesterol diets (1%-cholesterol diet + MK-329). The mucin content of the mucus layer on the epithelial surface of the gallbladder wall more than doubled (P < .05) with the high-cholesterol diet; adding MK-329 to the latter group produced a further 82% increase (P < .05). The cholesterol diet + MK-329 group had the highest (100%) incidence of cholesterol crystals that were evident in fresh gallbladder bile, coincident with a shortened nucleation time (2.5 +/- 0.6 days; P < .05 vs. the cholesterol diet + placebo group, 5.8 +/- 1.0 days or the other 2 groups, >21 days). Bile from animals on the trace-cholesterol diet, whether or not receiving MK-329, lacked crystals in bile and exhibited a normal nucleation time (>21 days). Thus, stasis per se may lower the CSI, but its detrimental effect on the gallbladder predominates locally, and so accelerates cholesterol crystal formation in this model.

The purpose of this study was to quantify and model various types of cell death for a small-cell lung cancer (SCLC) cell line (U1690) after exposure to a 137Cs source and as well as to compare the linear-quadratic (LQ) and repairable-conditionally repairable model (RCR). This study is based on four different experiments that were taken place at Cancer Centrum Karolinska (CCK). A human small-cell lung cancer (SCLC) cell line after the exposure to a 137Cs source was used for the extraction of the clonogenic cell survival curve. Additionally, for the determination and quantification of various modes of cell death the method of fluorescence staining was implemented, where the cell deaths were categorized based on morphological characteristics. The percentage of cells in each phase of the cell cycle was investigated with flow cytometry analysis. The quantification of senescent cells was performed by staining the samples with senescence-associated β-galactosidase (SA-β-Gal) solution and then scoring as senescent cells those that had incorporated the substance. These data were introduced into a maximum likelihood fitting to calculate the best estimates of the parameters used by the examined model. In this model, the modes of cell death are divided into three categories: apoptotic, senescent and other types of cell death (necrotic/apoptotic, necrotic, micronuclei and giant). In the clonogenic cell survival assay, the fitting of the RCR model gives a χ(2)-value of 6.10 whereas for the LQ model became 9.61. In the fluorescence microscopy and senescence assay, the probability of the three different modes of cell death on day 2 seems to increases with a dose up to about 10 Gy where there is saturation. On day 7 a significant induction of apoptosis in a dose- and time-dependent manner was evident, whereas senescence was slightly increased in response to dose but not to time. As for the 'other types of cell death' mode on day 7 showed a higher probability than the one on day 2 and as well as a prominent dose-dependence. The RCR model fits better to the experimental data than the LQ model. On day 2 there is a slight increase of the apoptotic and senescent probability with dose. On the other hand, on day 7 the shape of the curve of apoptosis differs and a sigmoidal increase with dose is observed. At both time-points, the present model fits the data reasonably well. Due to the fact that the clonogenic survival does not coincide with the one extracted from the fluorescence microscopy, a more accurate way to quantify cell death needs to be used, e.g. computerized video time-lapse (CVTL).

Dysfunction of human endothelial cells is an important trigger for atherosclerosis. Oxidative low-density lipoprotein (ox-LDL) usually was used to stimulate the dysfunction of human umbilical vein endothelial cells (HUVECs). LncRNA SNHG1 (small nucleolar RNA host gene 1) is a cerebral infarction-associated gene. The present study was designed to investigate the role of SNHG1 in ox-LDL-induced HUVECs. Cell viability was evaluated by CCK-8 and MTT assay. Cell apoptosis was detected by flow cytometry analysis. Cell inflammatory response was evaluated by detecting LDH, IL-6, IL-1β levels. The results revealed that up-regulation of SNHG1 attenuated ox-LDL-induced cell injury and inflammatory response in HUVECs. Next, mechanism assays including RNA immunoprecipitation (RIP) assay, luciferase reporter assay, and RNA pull-down assay, helped us to identify the interaction between miR-556-5 and SNHG1. GNAI2 (G protein subunit alpha i2) and PCBP1 (poly(rC) binding protein 1) were identified as the downstream targets of miR-556-5p. SNHG1 regulated dysfunctions of ox-LDL-induced HUVECs via sponging miR-556-5p and up-regulating GNAI2 and PCBP1. SNHG1 attenuated cell injury and inflammatory response in ox-LDL-induced HUVECs via up-regulating both GNAI2 and PCBP1 at a miR-556-5p dependent way.

Keywords: G protein subunit alpha i2; atherosclerosis; miR-556-5p; poly(rC) binding protein 1; small nucleolar RNA host gene 1.

OBJECTIVE

To investigate the anti-inflammatory effects of asiatic acid (AA) on lipopolysaccharide (LPS)-induced inflammatory response in human corneal epithelial cells (HCECs).

METHODS

Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA expression of interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β (TGF-β) in HCECs. Intracellular reactive oxygen species (ROS) was measured using the ROS assay kit. Glutathione (GSH) concentration was measured using the total GSH assay kit. Akt1 and Akt phosphorylation (p-Akt1) levels were measured by Western blotting and immunofluorescence.

RESULTS

AA induced toxicity at high concentrations and significantly stimulated the proliferation of HCECs at concentrations of 20 µmol/L for 1h. LPS at concentrations of 300 ng/mL for 1h significantly stimulated the mRNA expression of IL-8, IL-6, IL-1β, TNF-α, and TGF-β in HCECs, while the stimulation effects were significantly inhibited by AA (20 µmol/L). In addition, AA was found to decrease the content of ROS, increase GSH generation, and also inhibit LPS-induced p-Akt in HCECs.

CONCLUSIONS

AA decreases the generation of inflammatory factors IL-8, IL-6, IL-1β, TNF-α, and TGF-β in LPS-stimulated HCECs. AA significantly inhibites the intracellular concentrations of ROS and increases GSH generation. AA also inhibites LPS-induced p-Akt in HCECs. These findings reveal that AA has anti-inflammation effects in LPS-stimulated HCECs.

OBJECTIVE

To determine the cause of body weight loss after total gastrectomy.

METHODS

We evaluated the acute effect of exogenous cholecystokinin (CCK) on food intake and the chronic effect of CCK receptor blockade on food intake and body weight after total gastrectomy in rats.

RESULTS

Exogenous CCK significantly reduced food intake in gastrectomized rats; this was blocked by administration of a CCK-A but not a CCK-B receptor antagonist. Chronic treatment with a CCK-A or CCK-B receptor antagonist after total gastrectomy in rats significantly increased postoperative food intake and body weight.

CONCLUSIONS

Our data indicate that endogenous CCK is partly responsible for reduced food intake and body weight loss after total gastrectomy in rats.

The circadian rhythm of the body core temperature (T(c)) and the effects of changes in ambient temperatures on the homeostasis of T(c) in Otsuka Long Evans Tokushima Fatty (OLETF) rats, which are naturally occurring cholecystokinin (CCK)-A receptor (CCK-AR) gene knockout (-/-) rats, were examined. In addition, the peripheral responses to warming or cooling of the preoptic and anterior hypothalamic region (PO/AH) were determined. The circadian rhythm of T(c) in OLETF rats was similar to that in Long-Evans Tokushima (LETO) rats; this rhythm was characterized by a higher T(c) during the dark period and a lower T(c) during the light period. When the ambient temperature was changed within the limits of 0 degrees C to 30 degrees C, the changes in T(c) of LETO rats were associated with the changes in ambient temperature, whereas those in OLETF rats were dissociated from the temperature changes. The OLETF rats showed a large hysteresis. The peripheral responses to warming or cooling of PO/AH, including shivering of the neck muscle and changes in skin temperature of the tail and footpad, were similar in OLETF and LETO rats. To confirm the role of CCK-AR in the regulation of body temperature, the values of T(c) in the CCK-AR(-/-) mice were compared with those in CCK-B receptor (CCK-BR) (-/-), CCK-AR(-/-)BR(-/-), and wild-type mice. In the mice, the circadian rhythms of T(c) were the same, regardless of the genotype. Mice without CCK-AR showed larger hysteresis than mice with CCK-AR. From these results, we conclude that the lack of CCK-AR causes homeostasis of T(c) in rats and mice to deteriorate.

The importance of the infant's cholecystokinin (CCK) system for eliciting optimal maternal care was examined in 6-9-day-old Sprague-Dawley rats. After administration of either vehicle, CCK-8 (1 or 8 microg/kg) or devazepide (1 mg/kg; a selective CCK(A) receptor antagonist), pups were either individually isolated (Experiment 1) or individually reunited with their dam (Experiment 2) and the rats' behavior was observed. When isolated, pups that received devazepide displayed significantly more head-lifting and wall-climbing attempts than vehicle-treated controls, suggesting that endogenous CCK dampens activity. Devazepide-treated rats were found more frequently in proximity with their mothers when reunited with them, and they emitted more ultrasonic vocalizations compared to vehicle controls. Pups treated with 1 microg/kg CCK received less body licking than vehicle controls. In addition, dams hovered and crouched over devazepide-treated pups more than over pups treated with 1 microg/kg CCK. The results suggest that endogenous CCK has a calming, quieting effect in the neonatal pup and that this, in turn, results in less infant-mother attractivity and reduced levels of maternal care.

OBJECTIVE

To investigate the central regulation of food intake by quantifying neuron activation of the nucleus of the solitary tract (NTS) after injection of cholecystokinin (CCK) or food intake in gastrectomized rats.

BACKGROUND

Total gastrectomy is followed by early satiety, low calorie intake, and weight loss in the majority of patients. The etiology of these effects is unknown. Sixty percent to 70% of patients remain underweight after total gastrectomy, the weight loss averaging 25% of preoperative body weight. About two thirds of gastrectomized patients report early satiety, and about 60% do not reach the recommended daily calorie intake. The NTS is a brain stem center involved in the regulation of food intake; thus, the extent and pattern of neuronal activation provide information on the process involved in the initiation of satiation and the regulation of food intake.

METHODS

The authors investigated neuronal activation in the NTS using c-fos immunohistochemistry following CCK injection or food intake in healthy control rats, sham-operated control rats, age-matched control rats, weight-matched control rats, and vagotomized or gastrectomized rats.

RESULTS

Neuronal activation in the NTS after CCK injection was significantly decreased 21 days after total gastrectomy, but increased by up to 51% 3 months and by up to 102% 12 months after surgery compared to age-matched unoperated control rats. Neuronal activation in the NTS in response to feeding was markedly increased up to fivefold in gastrectomized rats. This increase was early in onset and sustained, and occurred despite significantly reduced food intake. Administration of MK329, a CCK-A receptor antagonist, significantly reduced the number of postprandially activated neurons in both gastrectomized and control rats.

CONCLUSIONS

The early postprandial activation of NTS neurons after total gastrectomy in rats may correspond to early satiety reported by patients, while the sustained activation of NTS neurons after a meal could contribute to a reduced daily calorie intake. These data suggest that a disturbed central regulation of food intake might contribute to early satiety, reduced food intake, and weight loss after total gastrectomy.

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.

As a group of intestinal hormones and neurotransmitters, cholecystokinins (CCKs) regulate and affect pancreatic enzyme secretion, gastrointestinal motility, pain hypersensitivity, digestion and satiety, and generally contain a DYMGWMDFG sequence at the C-terminus. Many CCKs have been reported in mammals. However, only a few have been reported in amphibians, such as Hyla nigrovittata, Xenopus laevis, and Rana catesbeiana, with none reported in urodele amphibians like newts and salamanders. Here, a CCK called CCK-TV was identified and characterized from the skin of the salamander Tylototriton verrucosus. This CCK contained an amino acid sequence of DYMGWMDF-NH2 as seen in other CCKs. A cDNA encoding the CCK precursor containing 129 amino acid residues was cloned from the cDNA library of T. verrucosus skin. The CCK-TV had the potential to induce the contraction of smooth muscle strips isolated from porcine gallbladder, eliciting contraction at a concentration of 5.0 x 10⁻¹¹ mol/L and inducing maximal contraction at a concentration of 2.0 x 10⁻⁶ mol/L. The EC50 was 13.6 nmol/L. To the best of our knowledge, this is the first report to identify the presence of a CCK in an urodele amphibian.

OBJECTIVE

Low concentrations of tumour necrosis factor-alpha (TNF-α) have been reported to promote osteogenic differentiation. In this study, a series of in vitro experiments was performed to investigate underlying molecular mechanisms involved.

METHODS

MC3T3-E1 murine preosteoblasts were treated with TNF-α at doses of 0, 0.1 or 1 ng/mL. The ephrinB2-EphB4 signalling pathway was activated using ephrinB2-fc, or inhibited using lentiviruses encoding siRNAs specifically targeting EphB4. Cell proliferation/survival was evaluated using the Cell Counting Kit-8 (CCK-8) assay, and expression levels of Runx2, BSP, ephrinB2 and EphB4 were determined using RT-PCR and Western blotting. ALP activity in these cells was also determined, and mineral nodule formation was evaluated with alizarin red S staining.

RESULTS

Low concentrations of TNF-α had no influence on cell proliferation/survival. However, expression levels of Runx2, BSP, ephrinB2 and EphB4, as well as ALP activity and mineral nodule formation, were significantly enhanced in MC3T3-E1 cells treated with low concentrations of TNF-α. Moreover, activation of the ephrinB2-EphB4 signalling pathway by ephrinB2-fc enhanced TNF-α-induced osteogenic differentiation, while down-regulation of EphB4 level reversed the positive effect of TNF-α.

CONCLUSIONS

Low concentrations of TNF-α promoted osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway.

Heart rate responses to i.v. administration of cholecystokinin-8 (CCK-8) were investigated in Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors and control Long-Evans Tokushima Otsuka (LETO) rats. The heart rate decreased after i.v. administration of 3 nmol.kg(-)(1) of CCK-8 in LETO rats, but not in OLETF rats. Bradycardia in the LETO rats disappeared after treatment with MK-329, but not after treatment with L-365,260. The expression of CCK-A receptor precursor mRNA was found exclusively in the atrium in LETO rats. These results suggest that CCK-8 decreases heart rate via CCK-A receptors located in the atrium of the rats.

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.

The receptor subtype mediating rat alpha-calcitonin gene-related peptide (alpha-CGRP)-induced inhibition of gastric emptying of a non nutrient solution was tested in conscious rats using the CGRP1 receptor antagonist, CGRP 8-37, and the CCK antagonist, MK-329. Intravenous injection of alpha-CGRP (0.5 micrograms) decreased gastric emptying to 26.5 +/- 5.8% from 46.4 +/- 3.9% in vehicle-treated group. Intravenous injection of CGRP 8-37 (15 micrograms) did not influence gastric emptying but completely prevented alpha-CGRP inhibitory effect whereas the 47% delay in gastric emptying induced by intravenous cholecystokinin-8 (CCK, 0.25 microgram) was not modified. The CCK antagonist, MK-329 (1 mg) reversed CCK- but not alpha-CGRP-induced delay in gastric emptying. These results demonstrate that CGRP 8-37 is a specific tool to block alpha-CGRP inhibitory action on gastric motor function and suggest that gastric stasis elicited by peripheral injection of alpha-CGRP may involve an interaction with a CGRP1 receptor subtype.