The anorectic actions of cholecystokinin (CCK)-8 and of a selective CCK-A agonist, A-71623, were examined in CD1 mice, beagle dogs, and cynomolgus monkeys. A-71623 suppressed intakes in all species tested, and the effects were blocked by a selective CCK-A antagonist, A-70104. In the dog only, CCK-8 was more potent on a molar basis compared to A-71623, although the effects of both CCK agonists were more short-lived in the dog compared to the other species tested. Our results support other evidence suggesting that the anorectic actions of exogenous application of CCK-8 in these species are mediated via stimulation of the CCK-A receptor subtype.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking cholecystokinin (CCK)-A receptors are hyperphagic and obese, and exhibit deficits in meal size control and in neuropeptide Y (NPY) gene expression in the dorsomedial hypothalamus (DMH). The present study was intended to determine whether these deficits would affect OLETF rat's response to an acute 24-h period of food deprivation. OLETF rats lost more body weight in response to deprivation but recovered their weight more quickly during refeeding than did lean Long-Evans Tokushima Otsuka (LETO) rats. Food deprivation decreased plasma glucose and leptin levels to a similar degree in both strains. Both groups increased intake during refeeding but the magnitude of increase was significantly greater in OLETF rats. Deprivation resulted in a significant elevation in arcuate NPY gene expression (approximately 47%) in LETO rats but only produced a small nonsignificant increase in the already decreased level of expression in OLETF rats (approximately 24%, P>.05). DMH NPY gene expression was not changed by deprivation in either OLETF or LETO rats. Although paraventricular corticotropin-releasing factor (CRF) expression was decreased by deprivation in LETO rats, CRF expression was not affected in OLETF rats. Together, these data suggested that OLETF rats lacking CCK-A receptors are not only capable of increasing their food intake in response to food deprivation, but also exhibit differential sensitivity to the effects of deprivation during both the food deprivation and refeeding periods.

The biochemical regulation of maternal behavior has been extensively studied. Cholecystokinin (CCK), a gut peptide that is also present in the brain, recently has been implicated in the onset of maternal behavior in estrogen-primed virgin rats. The objective of the present set of studies was to delineate the role of CCK in the onset (Experiments 1-3) and maintenance (Experiments 4 and 5) of maternal behavior in rats. In the first study intracerebroventricular (i.c.v.) administration of CCK was unable to stimulate the onset of maternal behavior in estrogen-primed virgin rats. Similarly, i.c.v. infusions of CCK into pregnant rats, starting on Day 17 of gestation (Experiment 2), did not advance the onset of maternal behavior. Moreover, when CCK-filled minipumps were implanted intraperitoneally in estrogen-primed virgin rats, the rate of onset of maternal behavior was unaffected (Experiment 3). In contrast, direct infusions of CCK into the MPOA blocked the disruptive effects of beta-endorphin on the maintenance of maternal behavior in postpartum lactating rats (Experiment 4). In addition, proglumide, a CCK receptor antagonist, disrupted maternal behavior in postpartum lactating rats by increasing latencies to retrieve and crouch over the young (Experiment 5). These results support an involvement of CCK in the maintenance, but not the onset, of maternal behavior in rats.

OBJECTIVE

Growth patterns of astrocytic tumors can be modulated in vitro by gastrin. In this study, the influence of gastrin on the in vitro cell cycle kinetics and the in vivo growth features of three experimental malignant gliomas was investigated.

METHODS

Gastrin-induced influence on overall growth was assayed in vitro by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium colorimetric assay for human U373 and rat C6 gliomas and for rat 9L gliosarcoma. Although cell cycle analyses were performed by means of computer-assisted microscope analyses of Feulgen-stained nuclei, the gastrin-induced effects of the levels of expression of cyclins D3 and E, CDK2, CDK4, CDK5, CDK7, p15, p16, E2F1, and E2F2 were assayed by means of quantitative Western blot test. The presence of ribonucleic acids for the CCK(B) and CCK(C) gastrin receptors in the U373, C6, and 9L models was assayed by means of quantitative reverse transcriptase-polymerase chain reaction, and the presence or absence of ribonucleic acids for CCK(A) receptor was checked by means of conventional polymerase chain reaction. The influence of gastrin was also characterized in vivo in terms of the survival periods of conventional rats orthotopically grafted with the C6 and 9L models and nude rats with the U373 model.

RESULTS

Gastrin significantly decreased the overall growth rate in the rat C6 and the human U373 high-grade astrocytic tumor models with either CCK(B) or CCK(C) gastrin receptor but not in the 9L rat gliosarcoma, which had no CCK(B) gastrin receptor (but had CCK(A) receptor) and only weak amounts of CCK(C) receptor. This effect seems to occur via a cytostatic effect; that is, an accumulation of tumor astrocytes occurs in the G(1) cell cycle phase. The cytostatic effect could relate to a gastrin-induced decrease in the amounts of the cyclin D3-CDK4 complex in both C6 and U373 glioma cells. In vivo, gastrin significantly increased the survival periods of C6 and U373 glioma-bearing rats, but not of 9L gliosarcoma-bearing rats.

CONCLUSIONS

Gastrin is able to significantly modify the growth levels of a number of experimental gliomas.

Impaired gallbladder emptying is frequent in cholesterol gallstone disease as well as in predisposing conditions like pregnancy and obesity. Gallbladder hypomotility is considered a pathogenic factor for gallstone formation, providing the residence time for cholesterol crystal nucleation, but any effect on the enterohepatic circulation of bile acids and subsequently on biliary lipid composition is unknown. Therefore, we studied the effect of prolonged suppression of gallbladder emptying with a cholecystokinin (CCK-A) receptor antagonist on bile formation in Richardson ground squirrels fed a trace versus a 1% cholesterol diet. Biliary lipid secretion was measured directly and bile acid pool size assessed by isotope dilution ([14C]-cholic acid). Gallbladder contraction was determined in vitro in response to CCK. The CCK-antagonist (MK-329) greatly inhibited gallbladder contraction in vitro and increased gallbladder fasting volume and bile acid pool size in vivo. It significantly lowered the cholesterol saturation index by 35% and 46% in hepatic bile and by 18% and 28% in gallbladder bile in the trace and cholesterol diet groups, respectively. Bile acid secretion and bile flow doubled with the CCK-receptor antagonist. Chronic CCK receptor antagonist-induced inhibition of gallbladder emptying increases bile acid secretion and thereby decreases cholesterol saturation in bile. Extensive biliary hypomotility thus leads to a more rapid cycling of bile acids by depriving the gallbladder of its function in the enterohepatic circulation.

Intraventricular cholecystokinin COOH-terminal octapeptide (CCK-8) decreases meal size in the meal-trained baboon. In the present study, we tested whether this action is mediated by CCK-A receptors, CCK-B receptors, or both. Intraventricular administration of the selective CCK-A receptor agonist AAdditionally, intraventricular ACCK-8 at 1 nmol/kg (% suppression of meal by CCK = 59 +/- 17). However, intraventricular administration of the CCK-B receptor agonist AAAACCK-8 treatment. Thus we conclude that the predominant receptor population with which intraventricular CCK-8 interacts are type-A CCK receptors that are accessible to the ventricular system of the baboon.

We examined the effect of troglitazone treatment on pancreatic growth in the CCK-A receptor-deficient Otsuka Long-Evans Tokushima fatty (OLETF) rat, an animal model for type 2 diabetes mellitus. A troglitazone-rich diet (0.2%) was given from 12 to 28 wk of age or from 12 or 28 wk of age to 72 wk of age. Fasting serum glucose concentrations in control OLETF rats increased progressively with age, which was almost completely prevented by troglitazone treatment. Insulin levels in serum and pancreatic content in the control rat markedly increased at 28 wk of age but significantly decreased at 72 wk of age compared with those at 12 wk of age, whereas those in troglitazone-treated rats were nearly the same at all ages and were similar to those in control rats at 12 wk of age. Pancreatic wet weight in control rats decreased with age irrespective of whether they were hyperinsulinemic (28 wk old) or hypoinsulinemic (72 wk old). Troglitazone treatment significantly increased pancreatic wet weight and protein, DNA, and enzyme contents compared with those in the control rats. Moreover, troglitazone treatment completely prevented or reversed histological alterations such as fibrosis, fatty replacement, and inflammatory cell infiltration. Our results indicate that troglitazone stimulates pancreatic growth in the congenitally CCK-A receptor-deficient OLETF rat not only by reducing insulin resistance and potentiating insulin action but also by suppressing inflammatory changes in the pancreas.

The gastric acid inhibitory activities of a peptide-like gastrin receptor antagonist, Boc-beta Ala-Trp-Leu-Asp-O(CH2)2-Ph-4-F (CH-486), a nonpeptide gastrin/CCK-B antagonist (L-365,260), and a CCK-A antagonist (L-364,718), were investigated in the gastric lumen-perfused anaesthetized rat. A single i.v. injection of CH-486, 100 mumol/kg, reduced acid secretion stimulated by pentagastrin, 15 micrograms kg/h, to unstimulated levels, with no recovery within 50 min. Histamine-, 0.1 mumol kg/min, and carbamylcholine-, 0.1 mg kg/h, stimulated acid secretion were also reduced to unstimulated levels by CH-486, 100 mumol/kg, although with these latter two stimulants the inhibition was transient. L-365,260 and L-364,718, 10 mumol/kg, significantly inhibited both pentagastrin- and histamine-stimulated acid secretion, the latter again transiently. We conclude that the non-selective nature of the gastric acid inhibitory activity of gastrin antagonists might allow novel approaches to control gastric acid secretion in peptic ulcer disease.

[3H]Propionyl-Tyr-(SO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2 ([3H]pBC 264) (98-100 Ci/mmol), a new peptidase-resistant cholecystokinin (CCK) agonist that is 1000-fold more potent for CCK-B than for CCK-A receptors, interacts, with a similar subnanomolar affinity, with a single class of binding sites (Kd, 0.15-0.2 nM) in brain membranes of mouse, rat, guinea pig, and cat, in Tris and Krebs buffers. The concentration of CCK-A receptors in rodent brain was estimated to be 8-10 fmol/mg of protein, by measurement of the Bmax values of the nonselective agonist [3H] propionyl-CCKCCK-B sites. In guinea pig and mouse brain, specific [3H]pBC 264 binding was not affected by NaCl and/or guanyl-5'-yl-imidodiphosphate. In contrast, in rat brain the affinity of [3H]pBC 264 was decreased and the maximal number of binding sites was increased by NaCl and the guanyl nucleotide or by alkaline treatment, suggesting that a proportion of CCK-B receptors are linked to guanine nucleotide-binding proteins. The Bmax of a CCKCCK-B antagonist L365,260 was 18-, 30-, and 64-fold less potent than [3H]pBC 264 in guinea pig, mouse, and rat, respectively. PD 134308, a CCK-B antagonist, was 20-fold less potent in rat brain than in guinea pig brain. Likewise, the cyclic analog BC 254 displayed a 30- and 60-fold lower affinity for mouse and rat than for guinea pig brain preparations. Together, these results suggest the presence of CCK-B receptor subtypes. In all tissues, the specific binding of [3H]pBC 264 at its Kd values was very high (75-90%) and higher than that of the hydrophobic CCK-B probe [3H]SNF 8702 (approximately 50%). Therefore, unlike [3H]SNF 8702, [3H]pBC 264 can be used to study preparations with low receptor concentrations, such as rat brain, making this radiolabeled molecule the most appropriate ligand available to date for CCK-B receptor studies.

OBJECTIVE

Bee venom (BV) has frequently been used as a remedy for inflammatory diseases. The aim of this study was to investigate the effect of BV on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis (AP) in rats.

METHODS

The BV pretreatment group: 0.25 mg/kg BV was administered subcutaneously, followed by 75 mug/kg CCK-8 subcutaneously 3 times after 1, 3, and 5 hours. This whole procedure was repeated for 5 days.

METHODS

CCK-8 subcutaneously 3 times after 1, 3, and 5 hours for 5 days. The BV posttreatment group: CCK-8 subcutaneously 3 times at an interval of 2 hours for 3 days, and then 0.25 mg/kg of BV was administered subcutaneously.

METHODS

CCK-8 subcutaneously 3 times at an interval of 2 hours for 3 days.

RESULTS

The BV pretreatment and posttreatment ameliorated many of the examined laboratory parameters (the pancreatic weight [PW]/body weight [BW] ratio, the serum amylase and lipase activity) and reduced histological damages in pancreas. Furthermore, BV pretreatment reduced the production of tumor necrosis factor-alpha, interleukin 1, and interleukin 6 and also decreased pancreatic nuclearfactor-kappaB binding activity compared with saline-treated group in the AP model. The BV also increased heat shock protein 60 (HSP60) and heat shock protein 72 (HSP72) compared with the saline-treated group in the AP model.

CONCLUSIONS

These findings suggest that the anti-inflammatory effect of BV in CCK-8-induced AP seems to be mediated by inhibiting nuclear factor-kappaB binding activity, and that BV may have a protective effect against AP.

Because of difficulties encountered in setting up radioimmunoassays for cholecystokinin (CCK) a sensitive and reliable biological method for estimating this hormone is still needed. The principles of such a biological technique and an improvement to it have already been described, but the serum levels of CCK reported were high and the technique required further refinement and validation. The strips of rabbit gall-bladder used to estimate the concentration of CCK increased in sensitivity to standard solutions of CCK over a 6--8 period before stabilizing, but a single sample of serum increased the sensitivity of the strips of gall-bladder to their maximum immediately. These two problems were eliminated by 'priming' the strips of gall-bladder by exposure to two serum samples before exposure to the standard solutions used for production of a dose--response curve. Thirdly, it was discovered that some non-peptide substances in serum possessed CCK-like activity; by extracting all the small peptides from serum with dextran-coated charcoal the residual activity could be measured and subtracted from the total CCK activity. Finally, the activity of CCK in the serum increased during processing before freezing. This increase was eliminated by taking the blood samples into aprotinin which has been shown to cause dramatic reduction in CCK activity in some experiments. When all these factors were taken into account and the technique suitably modified, the mean level of CCK in the serum of ten normal fasting subjects was found to be 28 milli Ivy Dog units/ml (2.4 pmol/ml), which is only one third of that reported previously.

Systemic injections of cholecystokinin (CCK), a "gut-brain" peptide, have been shown to modulate brain dopamine function and produce neuroleptic-like effects on such dopamine-regulated behaviors as locomotor activity. However, clinical trials of CCK agonists in schizophrenia patients showed mixed results. To re-examine the antipsychotic potential of CCK-based treatments, we examined systemic injections of CCK analogs in an animal model with strong face and construct validity for sensorimotor-gating deficits seen in schizophrenia patients and with strong predictive validity for antipsychotic drug activity. Prepulse inhibition (PPI) occurs when a weak acoustic lead stimulus ("prepulse") inhibits the startle response to a sudden loud sound ("pulse"). PPI is significantly reduced in schizophrenia patients and rats treated with dopamine agonists. Antipsychotics reverse decreased PPI in rats to a degree highly correlated with their clinical efficacy. Subcutaneous (s.c.) injections of caerulein (10 micrograms/kg) a mixed CCKA/B agonist, partially reversed amphetamine-induced reduction of PPI; whereas, s.c. haloperidol (0.5 mg/kg) totally reversed amphetamine-induced disruption of PPI. Caerulein's effect on PPI was blocked by pretreatment with a CCKA antagonist (devazepide) but not a CCKB antagonist (L-365,260). CCK-4, a preferential CCKB agonist, had no significant effect on PPI. These results suggest that caerulein produces a weak neuroleptic-like effect on PPI that is mediated by stimulation of CCKA receptors. Possible circuities in this effect are discussed. In a separate experiment, s.c. caerulein produced to a more potent neuroleptic-like profile on amphetamine-induced hyperlocomotion, suggesting that selection of preclinical paradigms may be important in evaluating the antipsychotic potential of CCK-based treatments.

The diphenylpyrazolidinone cholecystokinin (CCK)-B antagonist LY262691 has recently been demonstrated to decrease the number of spontaneously active dopamine (DA) cells in the ventral tegmental area (AACCK-B receptors, LY262684, LY191009 and LY242040, also decreased the number of spontaneously active AA cells. Neither an inactive analog (LY206890) nor a CCK-A-selective analog (LY219057) affected the number of spontaneously active AA cells. L-365,260, a benzodiazepine CCK-B antagonist, also decreased the number of spontaneously active AA cells. In addition, the more active optical isomer of LY262691 (LY288513) caused twice as large a decrease in the number of spontaneously active AA cells as the less active optical isomer (LY288512). The diphenylpyrazolidinone CCK-B antagonists, but neither the inactive nor the CCK-A selective analog, also decreased the number of spontaneously active AA cells; however, none of these compounds produced catalepsy in awake animals. Single-unit recordings indicated that LY262691 administration inhibited the activity of individual AAA neurons. These results indicate that the firing of AAA neurons is suppressed specifically by antagonism of CCK-B, but not CCK-A receptors. CCK-B antagonists may therefore represent a novel class of antipsychotic drugs. Furthermore, because CCK-B antagonists have no cataleptogenic effects, they may also have a reduced propensity for producing extrapyramidal side effects. In addition, these actions on midbrain DA neurons may contribute to the known anxiolytic activity of CCK-B antagonists.

The effect of dietary medium chain triglyceride (MCT) on short-term food intake was compared with the effect of long chain triglyceride (LCT) in rats. Corn oil and glyceryl tricaprylate were used as LCT and MCT sources, respectively. Rats were given diets containing 200 g MCT/kg diet (MCT diet), 100 g MCT + 100 g LCT/kg diet (ML diet), or 200 g LCT/kg diet (LCT diet) in Experiment 1. Cumulative food intake was determined every h for the first 12 h, then at 2-h intervals thereafter during the subsequent 12 h. As early as 1 h after feeding, cumulative food intake significantly decreased in MCT-fed animals in a dose-dependent fashion. In Experiment 2, rats were given a choice between MCT and LCT diets for 1 h to confirm whether or not the palatability of diets was influenced by dietary fat sources. There was no difference in food intake between the two diets. In Experiment 3, the responsibility of endogenous cholecystokinin (CCK) for the difference in food intake between the two diets was investigated for 6 h by using a CCK-A receptor antagonist, Devazepide (DVZ, 1 mg/kg b. wt.). Food intake in the MCT diet and also in the LCT diet was improved by DVZ. It is concluded that the satiety, but not the palatability, is affected by carbon chain length in dietary triglyceride sources, although the responsibility of endogenous CCK is very small.

Stress-induced analgesia (SIA) is an important endogenous mechanism in response to stressful stimuli. Opioid peptides, as well as endocannabinoids, are known mediators of SIA. We were interested in whether the endocannabinoid tone and the resulting SIA could be influenced by changing the activity of cholecystokinin (CCK) in mice. This hypothesis is related to recent evidence showing a cellular colocalization of neuropeptide CCK and cannabinoid type 1 (CB(1)) receptors in many central nervous system structures. However, the physiological relevance of this colocalization is unknown. Our aim was to test whether abolishing the endogenous CCK tone might influence the cannabinoid-mediated form of SIA. As expected, the CB(1) antagonist rimonabant prevented the development of analgesia in response to footshock-induced stress in wild-type mice. In contrast, CCK type 2 (CCK(2)) receptor-deficient mice developed SIA regardless of rimonabant treatment. Naloxone, an opioid antagonist, antagonized SIA in both wild-type and CCK(2) receptor-deficient homozygous mice. This finding suggests that CCK, through CCK(2) receptors, modulates the action of endocannabinoids. Gene expression analysis revealed an up-regulation of CCK(2) receptor gene in the lumbar spinal cord and mesolimbic area in wild-type mice in response to stress. In addition, wild-type mice displayed up-regulation of genes implicated in endocannabinoid-mediated neurotransmission (elevation of CB(1) receptor, 2-arachidonoylglycerol and the anandamide-synthesizing enzymes sn-1-diacylglycerol lipase alpha and N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D) in response to stress in the lumbar spinal cord and mesolimbic area. We did not find any of these changes in CCK(2) receptor-deficient homozygous mice. Altogether, behavioural and gene expression studies reflect an involvement of CCK(2) receptors in the development of endocannabinoid-mediated SIA.

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain. It is found in the highest levels in cortical and limbic structures and also in the basal ganglia. Two subtypes of CCK receptors have been described in the brain and gastrointestinal tissues. CCK(A) (alimentary subtype) receptors are mainly located in the gastrointestinal tract, regulating secretion of enzymes from the pancreas and emptying of the gallbladder. However, CCK(A) receptors are also found in several brain regions, with the highest densities in structures poorly protected by the haematoencephalic barrier (the area postrema, nucleus tractus solitarius and hypothalamus). The distribution of CCK(B) (brain subtype) receptors overlaps with the localisation of CCK and its mRNA in different brain areas, with the highest densities in the cerebral cortex, basal ganglia, nucleus accumbens and forebrain limbic structures.Both subtype of CCK receptor belong to the guanine nucleotide-binding protein-(G protein)-linked receptor superfamily containing 7 transmembrane domains. Signal transduction at CCK receptors is mediated via G(q) protein-related activation of phospholipase C and the formation of inositol 1,4,5-triphosphate (IP(3)) and 1,2-diacylglycerol (DAG). Recent cloning of CCK(A) and CCK(B) receptors has shown that mRNA for both receptors is distributed in the same tissues as established in radioligand binding and receptor autoradiography studies, with few exceptions.The existence of multiple CCK receptors has fuelled the development of selective CCK(A) and CCK(B) receptor antagonists. These antagonists belong to distinct chemical groups, including dibutyryl derivatives of cyclic nucleotides, amino acid derivatives, partial sequences and derivatives of the -COOH terminal sequence heptapeptides of CCK, benzodiazepine derivatives, 'peptoids' based on fragments of the CCK molecule, and pyrazolidinones. At the present time, the compounds of choice for blockade of the CCK(A) receptor are lorglumide, devazepide and lintitript (SR27897). L-365,260, CI-988, L-740,093 and LY288513 are the drugs most widely used to block CCK(B) receptors.Studies with CCK antagonists (and agonists) in animals and humans suggest a role for CCK in the regulation of anxiety and panic. The administration of CCK agonists [ceruletide (caerulein), CCK-4, pentagastrin] has an anxiogenic action in various animal models and in different animal species. However, the anxiogenic action of CCK agonists is restricted to nonconditioned (ethological) models of anxiety, with very limited activity in the 'classical' conditioned models. Pharmacological studies have revealed that CCK(B) receptors are the key targets in the anxiogenic action of CCK agonists. Nevertheless, CCK(B) antagonists displayed very little activity, if any at all, in these models, but strongly antagonised the effects of CCK(B) agonists. The anxiogenic/panicogenic action of CCK(B) agonists (CCK-4, pentagastrin) is even more pronounced in human studies, but the effectiveness of CCK(B) antagonists as anxiolytics remains unclear. Clinical trials performed to date have provided inconclusive data about the anxiolytic potential of CCK(B) receptor antagonists, probably because of limiting pharmacokinetic factors.The results of some animal experiments suggest a role for CCK in depression. The administration of CCK(B) antagonists causes antidepressant-like action in mouse models of depression. However, human studies replicating this result have yet to be carried out.A prominent biochemical alteration in schizophrenia is a reduction of CCK levels in the cerebral cortex. This change may be related to the loss of cortical neurons, due to the schizophrenic process itself. In animal studies (mainly in mice), administration of CCK agonists and antagonists has been shown to be effective in several models, reflecting a possible antipsychotic activity of these drugs. However, the data obtained in human studies suggest that CCK agonists and antagonists do not improve the symptoms of schizophrenia. Taking into account the reduced levels of CCK and its receptors found in schizophrenia, treatments increasing, but not blocking, brain CCK activity may be more appropriate.

Dihydroergotamine (dhe) (or phentolamine), an alpha-adrenergic blocking agent, induced important changes on the CCK-stimulated gallbladder emptying of 70 volunteer subjects. Two cholecystograms were performed with 10-day intervals in each subject. The first cholecystogram showed gallbladder emptying provoked by a test meal (35 subjects or by 0.5 U. CCK Kg. injected intravenously (35 subjects). During the second cholecystogram 1 mg. of DHE was injected intramuscularly 45 minutes befor the cholecystokinetic stimulus. The drug counteracted the gallbladder emptying induced by both endogenous and exogenous CCK. The effect was more pronounced when DHE was administered prior to the test meal stimulus than before CCK administration. This difference could be explained by a delayed gastric emptying induced by the alpha-adrenergic blockade. Our results suggest that the lack of gallbladder emptying could be due to the relaxation of this organ, in addition to a duodenal spasticity induced by DHE (or phentolamine).

The possible involvement of endogenous corticotropin-releasing factor (CRF) in the anxiogenic and pituitary-adrenal-axis-activating effects of cholecystokinin octapeptide sulfate ester (CCK 8) was investigated in rats. Intracerebroventricularly (i.c.v.) administered CCK 8 induced an anxiogenic response in an elevated plus-maze test, and enhanced the plasma corticosterone level. Pretreatment with different dilutions (1:10, 1:20 and 1:100, i.c.v.) of CRF antiserum and different doses of a CRF receptor antagonist, alpha-helical CRF (ahCRF, 0.001-1.0 microgram, i.c.v.) prevented the anxiogenic response to CCK 8 in a dose-dependent manner. None of the doses of CRF antiserum or ahCRF alone produced any alteration in either the elevated plus-maze paradigm or corticosterone level in saline-treated control rats. The results strongly suggest that the anxiogenic and hypothalamo-pituitary-adrenal-activating effects of CCK 8 are mediated via CRF.

We have cloned the mouse CCK-A receptor gene (Cckar), determined its nucleotide sequence, and analyzed its expression. The receptor protein is encoded in five exons distributed over 9 kb of genomic DNA. Intron/exon borders were determined by comparing the genomic nucleotide sequence with the mouse cDNA sequence obtained by reverse transcriptase polymerase chain reaction. RNase protection analysis of Cckar transcripts revealed the presence of a splice acceptor site 200 bp upstream of the translational start codon, indicating that the promoter is associated with a non-translated exon at an upstream site. The second coding exon contains a rarely used alternative splice site that would result in the production of a truncated, 48 amino acid protein. Cckar is widely expressed in the gastrointestinal system (pancreas, gallbladder, intestine, colon and stomach), as well as in brain and kidney.

Cholecystokinin (CCK) is a major gastrointestinal neuropeptide that is secreted in response to food ingestion. It is involved in the feedback regulation of gastric emptying and also modulates food intake. Leptin, a hormone that regulates food intake and energy balance, is secreted from adipose tissue, gastric mucosa, fundic glands, and other tissues. In a previous report we showed that gastric effects of leptin activated the nucleus tractus solitarius (NTS) neurons responding to gastric vagal stimulation. In this study, using the same in vitro neonatal rat preparation, we investigated the gastric effects of CCK and its interaction with leptin on NTS neurons receiving gastric vagal inputs. We observed that peripheral gastric effects of CCK (300 nM) produced a mean activation response of 271 +/- 3.9% compared with control level (100%) in 33 (60%) neurons tested (P <.01), and this response was abolished by a CCK-A receptor antagonist. A concentration-dependent effect of CCK (10 nM-1.0 microM) on NTS neuronal discharge frequencies was shown. We also observed that leptin (10 nM) applied to the stomach produced a mean activation response of 183 +/- 5.3% in 13 (50%) NTS units that responded to CCK (P <.01). Furthermore, we evaluated the combined effect of CCK and leptin in two groups of NTS neurons. Those NTS units that showed activation responses to both CCK (300 nM) and leptin (10 nM) had a subadditive effect that produced a mean activation response of 338 +/- 12.9% compared with the control level in all 10 (100%) neurons tested (P <.01). Eight (36%) of another 22 units that were not affected by either CCK (300 nM) or leptin (10 nM) alone had an activation response (151 +/- 5.2%; P <.05) when the same concentrations of CCK and leptin were applied together. Subsequently, by comparing the effects of CCK and leptin on a whole-stomach preparation to a partial-stomach preparation, we examined the area of the stomach in which gastric receptors contributed most to NTS unitary activity. We showed that the distal stomach containing the pylorus determined CCK gastric activity, whereas both the proximal and distal stomach are important for leptin's effect. Our data suggest that leptin modulates the potency of CCK signals that modify food intake in the neonatal rat.

We studied the behavioral effects of a novel cholecystokinin tetrapeptide (CCK-4) analogue, ACCK-A receptor subtype relative to the CCK-B receptor. In tests for anorectic activity, ACCK-A antagonist, ACCK-8, AACCK-8 diminished rapidly, whereas the suppressant effects of AACCK-A receptor agonist.

BACKGROUND

Whereas pepsin secretion by the human stomach has been extensively investigated, the characteristics of the peptide cell cannot be fully understood from in vivo studies. We therefore studied isolated human peptic cells to test directly cellular responses to different agents.

METHODS

Eight endoscopic biopsy specimens yielded 10(6) cells,>> 90% pure and>> 95% viable. Secreted pepsinogen was measured with a sensitive hemoglobin digestion method at pH 2.

RESULTS

Pepsinogen secretion was concentration-dependently stimulated by acetylcholine (ACh) (EC50 = 0.3 microM), cholecystokinin (CCK)-8 (2 nM), histamine (2 microM), and gastrin-I (30 nM) but not by bombesin or pentagastrin. ACh stimulation was inhibited 40 times more potently by atropine (IC50 = 12 nM) than by pirenzepine (IC50 = 0.5 microM). Histamine was inhibited by 10(-4) cimetidine. CCK-8 stimulation was inhibited 80% by the CCK-A-selective antagonist L364,718 (IC50 = 12 nM) but not by the CCK-B-selective antagonist L365,260.

CONCLUSIONS

Isolated human peptic cells from endoscopic biopsy specimens secrete pepsinogen in response to ACh>> CCK-8>> histamine>> gastrin-I. The human peptic cell muscarinic-cholinergic receptor is not of the M1 subtype, and the CCK-8 response is predominantly mediated by a CCK-A receptor subtype.

The effects of the partial muscarinic agonist pilocarpine on physiological responses were investigated in rat pancreatic acinar cells and compared with carbachol, a full muscarinic agonist, together with previous results using JMV-180, a partial agonist of CCK-A receptors. Pilocarpine was found to stimulate amylase release from isolated pancreatic acini in a concentration-dependent manner. At a maximal concentration (10 microM), pilocarpine was only capable of stimulating 63% of the secretion stimulated by a maximal concentration of carbachol. Moreover pilocarpine did not induce a decrease in secretion at supramaximal concentrations as does carbachol. In acini loaded with fura-2, superfusion of pilocarpine resulted exclusively in generation of intracellular Ca2+ concentration ([Ca2+]i) oscillations at all concentrations tested (0.3 microM-1 mM), in marked contrast to high concentrations of full agonists, which result in a biphasic sustained increase in [Ca2+]i. In common with low concentrations of other secretagogues that stimulate [Ca2+]i oscillations, pilocarpine at all concentrations was only able to stimulate a very small increase in phosphoinositide (PI) hydrolysis. In acini previously incubated with [3H]inositol, pilocarpine was shown to stimulate PI hydrolysis 27% above basal, compared with 872% for carbachol. To ascertain if this small degree of PI hydrolysis seen with pilocarpine is responsible for the generation of [Ca2+]i oscillations, an inhibitor of phospholipase C-linked processes, U-73122, which has been shown to inhibit Ca2+ oscillations induced by carbachol and CCK but not JMV-180 was tested. This agent rapidly inhibited pilocarpine-stimulated oscillations, indicating that in contrast to JMV-180, oscillations induced by pilocarpine are the result of PI hydrolysis.

OBJECTIVE

Cholecystokinin (CCK) is able to protect gastric mucosa against acute injury in experimental animals but little is known about the role of this hormone in maintaining mucosal integrity in humans. This double-blind, placebo controlled study was performed in 16 healthy volunteers. It describes the effects of CCK-8 infused intravenously (i.v.) at physiological doses and endogenous CCK released by intraduodenal (i.d.) oleate on gastric mucosal damage, as brought about by ethanol without or with pretreatment with loxiglumide, a selective CCK-A receptor antagonist.

METHODS

CCK-8 was infused i.v. 30 min before and throughout the study or i.d. oleate was instilled through a separate duodenal tube. Thirty minutes after the start of i.v. infusion of CCK or i.d. oleate instillation, 100 ml of 50% ethanol spray was applied to the gastric mucosa using an endoscope. Gastroscopy was performed and mucosal lesions were quantified using modified Lanza score. Gastric biopsies were taken from oxyntic mucosa for histological evaluation and gastric content was aspirated for radioimmunoassay of somatostatin.

RESULTS

In placebo-treated subjects ethanol caused endoscopic damage, with an average score of 2.8+/-0.2. Histologically, a widespread disruption of surface epithelium and deep hemorrhagic necrotic lesions were observed. Pretreatment with CCK or i.d. oleate markedly reduced the endoscopic lesion score to 0.7+/-0.1 and 0.3+/-0.1, respectively, and in both cases this reduction was accompanied by a significant rise in plasma CCK. Histologically, surface epithelium was still disrupted but deep necrotic lesions were absent. Gastric content collected before and after CCK or oleate showed a several-fold increase of luminally released somatostatin.

CONCLUSIONS

Pretreatment with loxiglumide abolished the protective effects of i.v. CCK-8 and i.d. oleate on mucosal lesions induced by ethanol and prevented the rise in intragastric somatostatin, but failed to affect the increments in plasma CCK. Endogenous CCK plays a physiological role in the maintenance of mucosal integrity. This occurs through activation of CCK-A receptors and is associated with an increased gastric luminal release of somatostatin.