OBJECTIVE

It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus.

METHODS

Canine CCK-58 was purified from intestinal extracts and CCK-8 was purchased. Their amounts were determined by amino acid analysis. The effect of tertiary structure on receptor binding at CCK-A receptors and CCK-B receptors was evaluated using membrane preparations from mouse pancreas and brain. The influence of C-terminal tertiary structure on stability to enzymatic digestion was evaluated by reacting CCK-8 and CCK-58 with endopeptidase 24:11.

RESULTS

CCK-58 was three times more potent than CCK-8 for binding mouse pancreatic membrane CCK-A receptors and equipotent to CCK-8 for binding mouse brain CCK-B receptors. CCK-8 was readily digested by endopeptidase 24:11, whereas CCK-58 was not.

CONCLUSIONS

The results strongly support the hypothesis that differences in tertiary structure of the carboxyl terminus of CCK-8 and CCK-58 influence receptor binding and stability to enzymatic digestion.

Previous biochemical characterizations of the cholecystokinin (CCK) receptor have used the "long" probe 125I-Bolton-Hunter-CCK-33 since it was the only CCK analogue with high affinity and high specific radioactivity which possessed an amino group available for chemical cross-linking. These studies have consistently identified a major binding protein of approximately 81 kilodaltons and have identified several minor proteins which were obtained under different cross-linking conditions and in different laboratories. Because the receptor-binding region of CCK-33 (carboxyl-terminal heptapeptide) is so far removed from the radiolabel and from available amino groups (positions 1 and 11), this probe carries potential for proteolytic cleavage of label from receptor and for labeling "near neighbors" instead of the binding site. We therefore designed two "short" probes for the CCK receptor. 125I-Bolton-Hunter-Lys-Gly-CCK-8 has an epsilon-amino group available for cross-linking. 125I-Tyr-[Thr28,Nle31]CCK-25-33 has an alpha-amino group for cross-linking and has the major advantage of being labeled by oxidative means, unique for CCK derivatives. Both radioiodinated decapeptides were purified by reverse-phase high pressure liquid chromatography to yield specific radioactivity of 2,000 Ci/mmol; demonstrated saturable, specific, and high affinity binding to rat pancreatic plasma membranes; and retained full biological activity to stimulate amylase secretion. Using a variety of cross-linking methods, these probes each identified the same Mr = 85,000-95,000 protein in rat pancreatic plasmalemma, and CCK-8 competed for this labeling in a concentration-dependent manner (IC50 = 1 nM). No change in apparent mobility of this band was observed under reducing or nonreducing conditions, suggesting lack of covalent attachment to other subunits. The Mr = 85,000-95,000 species migrated differently on sodium dodecyl sulfate gels than any of the components previously identified using 125I-Bolton-Hunter-CCK-33, confirming the novel nature of this binding protein. These short probes should be very useful for further characterization of CCK receptors on this and other tissues.

BACKGROUND

The effects of estradiol benzoate (EB) on gastric emptying, gastrointestinal transit and plasma levels of cholecystokinin (CCK) were studied in ovariectomized rats.

METHODS

Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal and Na2 51CrO4. Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Gastrointestinal transit was evaluated by calculating the geometric center of distribution of the radiolabeled marker. Blood samples were collected for E2 and CCK radioimmunoassay.

RESULTS

After treatment of EB (4-25 microg/kg), gastric emptying and gastrointestinal transit were inhibited, whereas plasma concentrations of E2 and CCK were increased in a dose-dependent manner. The selective CCK(A) receptor antagonists, devazepide and lorglumide, effectively attenuated the EB-induced inhibition of gastric emptying and gastrointestinal transit. L-365,260, a selective CCK(B) receptor antagonist, did not alter the EB-induced inhibition of gastric emptying and gastrointestinal transit.

CONCLUSIONS

The results suggest that EB inhibits gastric emptying and gastrointestinal transit in ovariectomized rats via a mechanism involving CCK stimulation and CCK(A) receptor activation.

Cholecystokinin (CCK) regulates intestinal motility after being released by several luminal nutrients. However the mechanism of action of CCK is still not well known. The aim of our study was to establish the mechanism of action of CCK in the rat intestine using an in vivo model and focusing on the nervous pathways involved in the response as well as type of receptors. Anesthetized rats were prepared with two strain-gauges, in duodenum and jejunum, to record circular muscle motor activity. A group of animals was also prepared with a catheter to infuse capsaicin inside the duodenum. Responses to CCK-octapeptide (CCK-8) as well as to CCK agonists were studied. CCK-8 was also infused after CCK antagonists, atropine, hexamethonium or L-nitroarginine. Results show that duodenal response to CCK-8 is excitatory although inhibitory responses can be induced by gastrin. In the jejunum, CCK-8 induces an inhibitory response that is mediated by both CCK-A and -B receptors. Excitatory responses to CCK-8 are due to stimulation of preganglionic receptors while inhibitory responses are NO mediated through stimulation of postganglionic CCK-B receptors. Capsaicin locally applied in duodenal mucosa significantly decreased CCK-8 response, whereas mucosal exposure to lidocaine completely blocked CCK-8 response. In conclusion our results show that CCK response varies along the intestine according to the predominance of excitatory or inhibitory efferent innervation. Moreover, CCK-8 actions are mediated through both extrinsic and intrinsic afferent fibres.

To find out whether physiological concentrations of cholecystokinin (CCK), a gastrointestinal hormone in mammals, are also active in chickens, the pancreatic amylase secretory response to CCK-8 was investigated in vitro. Rat pancreatic acini responded to the physiological concentration of CCK-8, but in chickens amylase release was induced at a concentration of CCK-8 1000 times higher than that observed in rats. In another experiment, biliary flow was tested with several doses of CCK-8. The bile flow was stimulated in a dose-dependent fashion, but a significant enhancement was not obtained at a concentration of 0.5 micrograms CCK-8/kg body weight, which was considerably higher than physiological ones. It is concluded that endogenous CCK does not have an important role in the digestive system in the chicken.

High concentrations of cholecystokinin (CCK) in the striatum and limbic areas of the brain suggest that this peptide may influence dopaminergic transmission. Thus, the effect of CCK on dopamine D2 receptors in the striatum and nucleus accumbens of the rat brain both in vitro and in vivo (central and peripheral administration) was studied by determining the binding of 3H-spiperone. The density (Bmax) of D2 receptors was elevated (a) by 20% in the accumbens upon in vitro co-incubation with 10(-6) M CCK. (A non-significant drop of 10% occurred in the striatum); (b) by about 40% in the accumbens and 25% in the striatum after continuous intraventricular infusion of CCK for 24 hr. The increase in receptor density in the accumbens was maintained for 14 days and in both tissues was specific to CCK (neurotensin infusion did not alter 3H-spiperone binding); (c) by 20% in the accumbens and 15% in the striatum 3 hr after a single IP injection of 50 micrograms/kg CCK or caerulein, and maintained up to 14 days later. These results suggest that CCK elevates dopamine D2 receptors in the accumbens and striatum and may be a physiological modulator of the dopaminergic system.

Recent studies have suggested that cholecystokinin (CCK) receptors may play a role in the development and growth of pancreatic cancers. We detected the expression of mRNA encoding CCK-A and CCK-B receptors in eight human pancreatic tumour cell lines using reverse transcription-polymerase chain reaction (RT-PCR), but not by RNase protection assays. The K-ras gene, which can be activated by G-coupled protein receptors such as CCK receptors, was mutated in codon 12 in five of the cell lines. In addition, Mia PaCa-2 pancreatic cancer cells did not respond to CCK or gastrin in cell proliferation or focal adhesion kinase (FAK) phosphorylation assays. In contrast, mouse NIH3T3 fibroblasts transfected with human CCK-B receptor (NIH3T3CCK-BR) showed increased proliferation and phosphorylation to the peptides. Also, radioligand binding studies indicated that Mia PaCa-2 cells had approximately 12.5-fold less CCK-B receptors than NIH3T3CCK-BR. Our results suggest that in Mia PaCa-2 cells, CCK receptors may not play a crucial role in supporting cell growth.

Using receptor binding assays, we have previously demonstrated the overexpression of the high-affinity cholecystokinin (CCK) receptor and the novel expression of the gastrin (CCK-B) receptor in the azaserine-induced rat pancreatic carcinoma DSL-6. Since cDNA of both the CCK-A receptor (classical pancreatic CCK receptor) coding region and the CCK-B receptor coding region have recently been cloned and sequenced, we investigated the expression of messenger RNA of these receptors in DSL-6 pancreatic carcinoma. Our results showed that the 32P-labelled cDNA probe of the CCK-A receptor coding region hybridized with an approximately 2.7 kb mRNA from both DSL-6 pancreatic carcinoma and normal rat pancreas. However, the relative expression of the CCK-A receptor mRNA in DSL-6 pancreatic carcinoma was approximately 8-fold of that in normal rat pancreas. The 32P-labelled cDNA probe of the CCK-B receptor coding region hybridized with an approximately 2.7 kb mRNA from DSL-6 pancreatic carcinoma; no hybridizing mRNA could be identified from normal rat pancreas. In summary, the CCK-A receptor mRNA is overexpressed approximately 8-fold and the gastrin (CCK-B) receptor mRNA is novelly expressed in DSL-6 pancreatic carcinoma as compared to normal rat pancreas. These results further confirm our previous findings based on receptor binding assays. The gene overexpression of the CCK-A receptor and the novel gene expression of the gastrin (CCK-B) receptor may be generated by alterations in gene regulation during carcinogenesis, and may play an important role in promoting tumor growth.

OBJECTIVE

To investigate the mechanism of the hypergastrinaemia associated with Helicobacter pylori infection by examining the effect of H. pylori infection and the cytokine tumour necrosis factor-alpha (TNF-alpha) on gastrin release from human antral fragments.

METHODS

In-vitro experimental study.

METHODS

Human antral biopsy fragments were cultured for 6 h with and without TNF-alpha (20 ng/ml) and the gastrin released over the following 2-h stimulation period measured by radioimmunoassay. The integrity of the paracrine feedback loop inhibiting gastrin release was tested by concurrent administration of cholecystokinin (CCK).

RESULTS

H. pylori-positive fragments were associated with significantly greater bombesin-stimulated gastrin release (increased by 40%, P < 0.05) and less inhibition produced by CCK administration (decreased by 55%, P < 0.05), than H. pylori-negative fragments. TNF-alpha treatment of H. pylori-negative fragments significantly enhanced bombesin-stimulated gastrin release (by 82%, P < 0.01) and diminished inhibitory feedback by CCK (by 53%, P < 0.05).

CONCLUSIONS

H. pylori infection is associated with enhanced gastrin release from human antrum and TNF-alpha produces a similar effect. Proinflammatory cytokines generated in the antrum in response to the infection are likely to play a significant role in the hypergastrinaemia of H. pylori infection.

To explore the mechanism underlying cholecystokinin-octapeptide (CCK-8) induced attenuation of pulmonary arterial hypertension (PAH) in endotoxic shock, the effects of CCK-8 on the changes in rabbit pulmonary arterial reactivity induced by tumor necrosis factor-alpha (TNF-alpha) were observed with the isolated arterial ring technique, and the ultrastructure of pulmonary arterial endothelium was observed under a scanning electron microscope. The contractile response to -adrenoceptor agonist phenylephrine (PE), the endothelium-dependent relaxation response to acetylcholine (ACh) and the endothelium-independent relaxation response to sodium nitroprusside (SNP) were not affected by TNF-alpha (4000 U/ml) after incubation for 2 h, while, if the incubation time was prolonged to 7 or 14 h, the relaxation response of pulmonary artery to ACh was depressed significantly, which, however, could be reversed by concomitant exposure to CCK-8 (0.5 microgram/ml). Incubation of pulmonary artery with CCK-8 (0.5 microgram/ml) alone did not bring out any contractile responses. Moreover, CCK-8 (0.5 microgram/ml) alleviated the ultrastructural lesions induced by TNF-alpha (4000 U/ml). These results suggest that CCK could protect pulmonary arterial endothelium against the detrimental effects by TNF-alpha.

Effects of C-terminal octapeptide of cholecystokinin (C8-CCK) and prostaglandins E1, E2 and E alpha on noradrenaline-induced responses and 3H-noradrenaline release in the gallbladder and sphincter of Oddi of guinea pigs were examined. In the sphincter of Oddi, noradrenaline in low concentrations induced a relaxation which was blocked by either phentolamine or propranolol, while noradrenaline in high concentrations induced a contraction which was blocked by phentolamine. These results suggest the existence of excitatory and inhibitory alpha-receptors and inhibitory beta-receptors in the sphincter of Oddi. In the gallbladder, the adrenergic receptors are alpha-excitatory and beta-inhibitory. C8-CCK (10(-9) g/ml) potentiated both contractile and relaxing responses to noradrenaline, in the gallbladder. The same concentration of prostaglandins potentiated only contractile response to noradrenaline. In the sphincter of Oddi, noradrenaline-induced responses were not affected by C8-CCK and prostaglandins. Prostaglandins inhibited 3H-efflux evoked by electrical stimulation, while C8-CCK had no effect on the 3H-efflux from both preparations. These results suggest that C8-CCK enhances the contractile and relaxing responses to noradrenaline, and that prostaglandins act in a similar way on the postsynaptic response and, in addition, inhibit presynaptically the release of noradrenaline in the gallbladder. In the sphincter of Oddi, only prostaglandins inhibit the presynaptic event.

The responsiveness of cultured myenteric neurons to cholecystokinin (CCK-8) was examined using fura-2-based digital microfluorimetric measurement of intracellular calcium ([Ca(2+)](i)). CCK-8 (10(-10)-10(-6)M) evoked concentration-dependent increases in percentage of neurons responding (8-52%) and delta[Ca(2+)](i) (76-169 nM). Gastrin (1 microM) also induced an increase in [Ca(2+)](i) in 29+/-6% of neurons (delta[Ca(2+)](i): 71+/-3 nM). L-364,718, an antagonist for the CCK-A receptor, blocked [Ca(2+)](i) response to CCK-8. Removal of extracellular calcium eliminated CCK-induced [Ca(2+)](i) increments, as did the addition of the calcium channel inhibitors nickel (1mM) and lanthanum (5mM). Nifedipine (1-50 microM) dose-dependently attenuated CCK-caused [Ca(2+)](i) responses. CCK evokes [Ca(2+)](i) signaling in myenteric neurons by the influx of extracellular calcium, likely through L-type calcium channels.

The influence of the cholecystokinin (CCK)A receptor antagonist, L-364,718, on beta-cell activation was examined in isolated perifused prelabelled rat islets. Insulin secretion and 3H efflux from myo-[2-3H]inositol-prelabelled islets (reflecting phosphoinositide hydrolysis) stimulated by CCK-8 (100 nM) were both inhibited by L-364,718, partially at 1 nM and totally at 10 nM. 45Ca2+ efflux from prelabelled islets was markedly stimulated by CCK-8. This stimulation was inhibited equally by 1 and 10 nM L-364,718. CCK-8 stimulated the 86Rb+ efflux (reflecting K+ movements) from prelabelled islets, which probably reflects an indirect effect of CCK-8 due to opening of Ca(2+)-activated K+ channels. This 86Rb+ efflux was inhibited by L-364,718 at 10 nM but not affected by L-364,718 at 1 nM. It is concluded that insulin secretion, phosphoinositide hydrolysis, Ca2+ and K+ movements stimulated by CCK-8 in isolated islets are all events mediated by CCKA receptors. The L-364,718-induced inhibition of phosphoinositide hydrolysis was most closely correlated to the inhibition of insulin secretion. This suggests that induction of cellular events activated through stimulation of phosphoinositide hydrolysis is a major mechanism underlying CCK-8-stimulated insulin secretion.

Central regulation of energy balance in seasonal mammals such as the Siberian hamster is dependent on the precise integration of short-term satiety information arising from the gastrointestinal tract with long-term signals on the status of available energy reserves (e.g. leptin) and prevailing photoperiod. Within the central nervous system, the brainstem nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN) are major relay nuclei that transmit information from the gastrointestinal tract to higher forebrain centres. We extended studies on the seasonal programming of the hypothalamus to examine the effect of the photoperiod on neuropeptidergic circuitries of this gut-brain axis. In the NTS and PBN we performed gene expression and immunoreactivity (-ir) studies on selected satiety-related neuropeptides and receptors: alpha-melanocyte stimulating hormone, melanocortin-3 receptor, melanocortin-4 receptor (MC4-R), growth hormone secretagogue-receptor, cocaine- and amphetamine-regulated transcript, preproglucagon (PPG), glucagon-like peptide 1 (GLP-1), cholecystokinin (CCK), peptide YY, galanin, neurotensin, and corticotrophin releasing hormone (CRH). Gene expression of PPG and MC4-R, and -ir of CCK and GLP-1, in the NTS were up-regulated after 14 weeks in long-day photoperiod (16 h light:8 h dark) compared to short-days (8 h light:16 h dark), whereas CRH-ir and NT-ir were increased in short-days within the PBN. We suggest that brainstem neuroendocrine mechanisms contribute to the long-term regulation of body mass in the Siberian hamster by a photoperiod-related modulation of satiety signalling.

Little attention has been paid to a possible relationship between lysosomal function and stimulation of secretory processes in endocrine cells. The last few years it has become increasingly evident that the secretion of insulin from the pancreatic beta-cell is the result of a very complex cascade of events, the details of which are far from elucidated and indeed may include the participation of the lysosomal system. We report here, with a combined in vitro and in vivo approach, that selective inhibition of islet lysosomal glycogenolytic acid glucan-1,4-alpha-glucosidase activity by the long-acting 1-deoxynojirimycin derivative emiglitate induces a profound suppression of nutrient-induced insulin release. In islet homogenate emiglitate strongly and dose-dependently inhibited the activity of acid glucan-1,4-alpha-glucosidase (EC50 approximately 10(-6) M) without affecting other classical lysosomal enzyme activities. The emiglitate-induced inhibition curve for glucose-stimulated insulin secretion from isolated islets was remarkably similar to the inhibition curve for acid glucan-1,4-alpha-glucosidase. Moreover, insulin release stimulated by the nonglucose nutrient secretagogues, leucine, and alpha-ketoisocaproic acid (KIC) was totally suppressed by emiglitate. In contrast, receptor activated insulin secretion induced by the insulinotropic hormone cholecystokinin (CCK-8) was unaffected by the drug. Further, parenteral pretreatment of mice with emiglitate markedly suppressed the insulin secretory response to an iv injection of glucose or KIC, whereas the response to an iv injection of CCK-8 was unaffected. In accordance with this, islets isolated from emiglitate-treated mice showed a reduced activity of acid glucan-1,4-alpha-glucosidase and, moreover, such islets incubated in vitro, secreted less insulin in response to glucose than did control islets. Finally, pretreatment of mice with purified fungal acid glucan-1,4-alpha-glucosidase, enzyme replacement, brought about a markedly increased insulin secretory response after an iv injection of KIC, whereas the insulin response after CCK-8 injection was unaffected. Taken together with previous observations, the present data strongly suggest that islet lysosomal acid alpha-glucosidehydrolases are involved in the multifactorial process of nutrient-induced insulin secretion. The existence of hitherto unresolved and complex interactions between different beta-cell organelles in the insulin secretory processes should be thoroughly reevaluated.

We have found that jejunal infusions of long-chain fatty acids, linoleic acid (LA) and oleic acid (OA), and gastric infusions of a fatty acid ethyl ester, ethyl oleate (EO), produce long-lasting suppression of total caloric intake. This effect is not seen in response to jejunal infusions of medium-chain fatty acids or medium- or long-chain triglycerides. Multiunit recordings have shown that intestinal infusions of LA or OA strongly activate celiac vagal afferents. Truncal vagotomy (TVX) and selective celiac-branch vagotomy (CVX) are equally effective in attenuating, but not eliminating, suppression of food intake by LA and EO. These outcomes suggest that intraintestinal fatty acids reduce intake by activation of vagal mechanisms, critically involving afferent fibers within the celiac branches, as well as unidentified nonvagal mechanisms. The role of cholecystokinin (CCK) in mediating the activation of celiac vagal afferents is suggested by studies showing that (1) inhibition of food intake by CCK-8 administration is attenuated after CVX but robust after celiac-spared vagotomy (CSV), (2) multiunit activity of celiac vagal afferents is increased by CCK-8 administration, and (3) activation of celiac fibers by intestinal LA infusion is severely attenuated by the CCK(A) antagonist lorglumide.

The ability to make repetitive non-invasive measurements of gastric emptying of nutritive solids in awake, unstressed mice is highly desirable. The aim of the present study was to develop such a technique using nuclear scintigraphy and diets differing in triglyceride content. Awake mice were accustomed to light restraint and to feeding cooked, egg white (0.00 g fat g(-1)), whole egg (0.10 g fat g(-1)), or egg yolk (0.31 g fat g(-1)). Gastric emptying of each diet was measured by labelling the test meals with Technetium(99m) Mebrofenin and using a conventional gamma camera equipped with a high resolution, parallel hole collimator. Gastric emptying of cooked whole egg was also determined following administration of either vehicle or CCK A receptor antagonist, devazepide. The half-emptying time (t(1/2)) significantly increased with increasing triglyceride content from 14 +/- 5 min to 51 +/- 6 min and 82 +/- 4 min for egg white, whole egg and egg yolk, respectively. Administration of devazepide significantly decreased t(1/2) of whole egg to 28 +/- 2 min. These results demonstrate the sensitivity and predictability of this technique in mice and importantly, provide an opportunity to alter the macronutrient or caloric content of the meal to determine effects on gastric emptying.

Several peroxisome proliferators have been shown to produce pancreatic acinar cell hyperplasia/adenocarcinomas in 2-year bioassays with rats: ammonium perfluorooctanoate (C8), clofibrate, methylclofenapate, HCFC-123, and Wyeth-14,643 (WY). We have used in vitro (C8, WY) and in vivo (WY) approaches to examine several possible mechanisms of pancreatic tumorigenesis by peroxisome proliferating compounds. These mechanisms include cholecystokinin receptor agonism (CCK(A)), trypsin inhibition, alterations in gut fat content, cholestasis, and altered bile flow/composition. All of these mechanisms enhance pancreatic growth either by binding to the CCK(A) receptor or by increasing plasma CCK levels. In vitro experiments using a receptor competition binding assay demonstrated that WY and C8 do not bind directly to the CCK(A) receptor. In a continuous spectrophotometric assay, WY and C8 also failed to inhibit trypsin, a common mechanism for increasing plasma CCK levels. These in vitro results suggested that WY was not acting via the two most common mechanisms for modulation of pancreas growth. Two types of in vivo experiments were conducted. The subchronic study (2-month duration) was designed primarily to detect early changes in pancreatic growth such as those mediated by compounds that inhibit trypsin or act as CCK(A) receptor agonists. The chronic study (6 months) was designed primarily to evaluate whether the pancreatic lesions were secondary to hepatic changes such as cholestasis and/or altered bile flow/composition. In the in vivo experiments, male Crl:CDBR rats were fed diets containing 0 or 100 ppm WY. In the subchronic study WY-treated rats had a twofold increase in mean relative liver weights, an eightfold increase in hepatic peroxisomal proliferation, and a fourfold increase in hepatocyte cell proliferation after 1 week which remained elevated throughout the 2 months of treatment. In contrast, no pancreatic weight effects, increases in plasma CCK, or acinar cell proliferation was seen through 2 months in the WY group when compared to the control group. Fecal fat concentrations were also measured at 2 months and demonstrated no difference between control and WY-treated animals. The absence of any early pancreas changes in the subchronic study is consistent with the in vitro data which demonstrated that WY is not a CCK(A) agonist or a trypsin inhibitor. The chronic study demonstrated increases in pancreatic weights at 3 months (6% above control) and 6 months (17% above control), as well as increased CCK plasma levels in the WY-treated group. Liver effects in the chronic study paralleled those of the subchronic time points. Clinical pathology endpoints including increased serum concentrations of bile acids, alkaline phosphatase, and bilirubin were indicative of cholestasis in the chronic WY-treated group. The cholestasis may be responsible for the downward trend in total bile acid output, both of which may contribute to the modest increases in plasma CCK levels. These results indicate that chronic exposure to WY causes liver alterations such as cholestasis, which may increase plasma concentrations of CCK. Hence, WY may induce pancreatic acinar cell adenomas/adenocarcinomas via a mild but sustained increase in CCK levels secondary to hepatic cholestasis.

Little is known as yet about the role of apoptosis in pancreatic damage. This study evaluated the effects of supraphysiologic concentrations of the cholecystokinin (CCK) analog, cerulein, which causes cell damage in vitro and acute pancreatitis in vivo, on cell proliferation and DNA fragmentation in the rat pancreatic acinar cell line AR4-2J. Cerulein inhibited the cell proliferation of AR4-2J time- and dose-dependently to approximately 60% of the control level at 10(-6) M after 72 h. DNA fragmentation, as assessed by both electrophoresis and enzyme-linked immunosorbent assay (ELISA), occurred at cerulein concentrations>> or = 10(-8) M. The maximal DNA fragmentation as measured by ELISA was reached after 24 h. Cerulein at concentrations>> or = 10(-9) M induced wild-type p53. Glutathione (1 mM) diminished the effects of cerulein on both cell proliferation and DNA fragmentation, whereas spermine (100 microM), which partially attenuated DNA fragmentation, did not have an effect on cell proliferation. The CCK-A-receptor antagonist loxiglumide completely abolished the effect of cerulein on DNA fragmentation. The serine-protease inhibitor FUT-175 (10 microM), the cysteine-protease inhibitor NCO-700 (5 mM), and ethylene glycol tetraacetic acid (EGTA; 500 microM) all had no effects on the changes in cell proliferation and DNA fragmentation induced by cerulein. The data suggest that supraphysiologic concentrations of cerulein rapidly induce apoptosis in AR4-2J cells and only later inhibit cell proliferation. These effects are mediated by CCK-A receptors. Cerulein-induced apoptosis may involve the induction of wild-type p53 or glutathione depletion or both.

Several studies have demonstrated that administration of cholecystokinin (CCK) reduces food intake in several species, including humans. In animal studies CCK-receptor antagonists have been reported to increase food intake, suggesting a physiological satiety effect of CCK in these animals. In a double-blind, placebo-controlled study, we investigated the effect of the specific CCK-A receptor antagonist loxiglumide on food intake (carbohydrate-rich meal) and on subjective hunger feelings scored with visual analogue scales and food selection lists in seven healthy obese women and in seven healthy lean women. Loxiglumide was administered intravenously in a dose of 10 mg/kg ideal weight/h. For the whole group, food intake during loxiglumide (359 +/- 39 g) was not significantly different from food intake during saline infusion (333 +/- 31 g). Also, when the lean and obese subgroups were analyzed separately, no significant influence of loxiglumide on food intake was found. In addition, no significant differences in satiety scores were seen using the food selection lists or visual analogue scales. In conclusion, in the present study during infusing the CCK-A receptor antagonist loxiglumide we found no increase in preprandial satiety nor in food intake of a carbohydrate-rich meal nor in postprandial satiety in lean and obese women.

Naloxone, added after contractions induced by CCK-8 on the guinea pig ileum preparation, elicited a contraction attributed to the release of endogenous opioid which could inhibit the excitatory action of the peptide. With large concentrations of CCK-8, the preparation gave reproducible responses with time. Naloxone, added before the peptide, protracted the excitatory response to CCK-8, but not its height. Morphine decreased the response to CCK-8 but simultaneously raised the response to naloxone. The latter effect appeared very similar to the withdrawal contraction observed after brief exposure of the opioid in the guinea pig ileum to opioids. Clonidine, and alpha-2 adrenoceptor agonist, and nifedipine, a calcium channel antagonist, both known to interfere with tolerance and physical dependence, affected the excitatory response to CCK-8 and the subsequent response to naloxone in a different way.

In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors L-NAME and alpha-guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCK-induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHP-2 mutant and the betaARK1 (495-689) sequestrant peptide, indicating the requirement of activated SHP-2 and G-betagamma subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHP-2 activity) revealed that in response to CCK-treatment, SHP-2 associated to G-beta1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.

The role of CCK in mediating neuronal activity in the brain in response to dietary carbohydrate was measured by detecting Fos immunoreactivity in response to duodenal glucose load in rats after administration of the CCK-A receptor antagonist devazepide. In adult, male Sprague-Dawley rats, infusion for 30 min of 545 mg (2.18 kcal) dextrose through a duodenal cannula induced Fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), lateral division of the central nucleus of the amygdala (CeAL), and the external subnucleus of the lateral parabrachial nucleus (LPBE). Devazepide treatment (1 mg/kg) attenuated Fos expression in the NTS and AP by 81 and 78%, respectively, but not in the CeAL or LPBE. These results indicate that central neuronal activation is elicited by dietary glucose in the intestinal lumen and that activation of neurons in the NTS and AP is mediated by CCK-A receptors.

The jejunal inflammation induced in rats by the nematode Nippostrongylus brasiliensis is followed by intestinal neuroimmune alterations including mast cell hyperplasia and nerve remodelling. On the other hand, cholecystokinin (CCK) plays a pivotal role in the regulation of intestinal motility. The aim of this study was to determine whether the intestinal motor response to CCK is altered 30 days after infection by N. brasiliensis. Thus, CCK-8 (50 microg kg(-1) intraperitoneally) disrupted the pattern of jejunal migrating myoelectric complexes for a longer time in postinfected rats (95.5 +/- 3.5 min) than in controls (48.1 +/- 5.1 min). This enhanced jejunal response was also found after oral administration of the potent releaser of endogenous CCK, soybean trypsin inhibitor. In contrast, no alteration of the inhibition of colonic motility by CCK administration was observed. The increased responsiveness of jejunal motility to CCK persisted after mast cell stabilisation or depletion but was prevented by atropine, devazepide and L-365260 (CCK-A and CCK-B receptor antagonists, respectively) and vagotomy. These results indicate that neuroimmune alterations after N. brasiliensis infection lead to an increased intestinal motility response to CCK that involves a cholinergic mediation, a vagal pathway and alterations in intestinal CCK-A and CCK-B receptors.