Inflammatory mediators are involved in the early phase of acute pancreatitis, but the cellular mechanisms responsible for their generation within pancreatic cells are unknown. We examined the role of nuclear factor-kappaB (NF-kappaB) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokine expression in pancreatic acinar cells in vitro. Supraphysiological, but not physiological, concentrations of CCK-8 increased inhibitory kappaB (IkappaB-alpha) degradation, NF-kappaB activation, and mob-1 gene expression in isolated pancreatic acinar cells. CCK-8-induced IkappaB-alpha degradation was maximal within 1 h. Expression of mob-1 was maximal within 2 h. Neither bombesin nor carbachol significantly increased mob-1 mRNA or induced IkappaB-alpha degradation. Thus the concentration, time, and secretagogue dependence of mob-1 gene expression and IkappaB-alpha degradation were similar. Inhibition of NF-kappaB with pharmacological agents or by adenovirus-mediated expression of the inhibitory protein IkappaB-alpha also inhibited mob-1 gene expression. These data indicate that the NF-kappaB signaling pathway is required for CCK-8-mediated induction of mob-1 chemokine expression in pancreatic acinar cells. This supports the hypothesis that NF-kappaB signaling is of central importance in the initiation of acute pancreatitis.

Iloperidone is a novel psychotropic compound currently undergoing Phase III trials. Its affinity for human dopamine and 5-HT(2A) and 5-HT(2C) receptors has been reported previously. This report presents the affinity of iloperidone for a largely extended number of human neurotransmitter receptors. In a few instances human receptors were not available and receptor studies were performed on tissues from laboratory animals. The present data, supplemented with those of, indicate that iloperidone displays high affinity (K(I) < 10 nM) for norepinephrine alpha(1)-adrenoceptors, dopamine D(3) and serotonin 5-HT(2A) receptors. Intermediate affinity (10-100 nM) was found for norepinephrine alpha(2C)-adrenoceptors, dopamine D(2A) and D(4) receptors and serotonin 5-HT(1A), 5-HT(1B), 5-HT(2C) and 5-HT(6) receptors. The affinity for all other receptors was below 100 nM, including norepinephrine alpha(2A), alpha(2B), beta(1), and beta(2), muscarine M(1)-M(5), histamine H(1), dopamine D(1) and D(5), CCK(A) and CCK(B), 5-HT(7), dopamine and norepinephrine transporters. Thus, iloperidone targets a selective set of dopamine, norepinephrine and serotonin receptor subtypes. The affinity for this particular set of receptors indicates that iloperidone has the potential to be a broad spectrum antipsychotic, with efficacy against positive, negative, depressive and cognitive symptoms of schizophrenia, and a low propensity to induce side effects.

Exogenous prostaglandins (PG) applied in small gastroprotective doses fail to affect healing of gastro-duodenal ulcers but accelerate the healing when used in larger gastric inhibitory doses that appear to enhance COX-2 expression and PGE(2) generation in the ulcer area. COX-1 and COX-inhibitors delay ulcer healing, particularly when both COX isoforms are suppressed such e.g. by indomethacin. Dexamethasone, that decreases the expression of COX-2 and mucosal generation of PGE(2), delays ulcer healing that can be reversed by the addition of small dose of exogenous PGE(2). Proton pump inhibitors (PPI) such as omeprazole and PGE analogs, accelerate ulcer healing mainly due to potent inhibition of gastric acid secretion, but they also augment the COX-2 expression and enzyme activity in the ulcerated mucosa. Endogenous PG generated at ulcer margin appear to be involved in ulcer healing promoted by growth factors and gut hormones such as gastrin or CCK and melatonin acting, at least in part, through increase of induction of COX-2 and local release of PGE(2) in the ulcer area. The ulcer healing activity of growth factors (e.g. EGF, TGF alpha, HGF) and certain gut hormones (gastrin, CCK) as well as melatonin, can be attenuated by treatment with COX-1 or COX-2 inhibitors which suppress the release of PGE(2) but enhance the expression of COX-2. It is concluded that endogenous PG originating mainly from upregulated COX-2 at the ulcer margin play crucial role in ulcer healing by exogenous PG, PPI, growth factors, gut hormones and melatonin, while COX-1 and COX-2 inhibitors delay ulcer healing by suppressing PG generation, and increasing COX-2 expression in the ulcer area.

In vitro autoradiography was performed in order to visualize cholecystokinin-A (CCK-A) receptors in sections of Cynomolgus monkey brain. CCK-A receptors were defined as those which displayed high affinity for the selective non-peptide antagonist MK-329 (L-364,718) and were detected in several regions by selective inhibition of 125I-Bolton Hunter CCK using MK-329 or direct labeling with 3H-MK-329. In the caudal medulla, high densities of CCK-A sites were present in the nucleus tractus solitarius, especially the caudal and medial aspects, and also the dorsal motor nucleus of the vagus. CCK-A sites were localized to a number of hypothalamic nuclei such as the supraoptic and paraventricular nuclei, the dorsomedial and infundibular nuclei as well as the neurohypophysis. The mammillary bodies and supramammillary nuclei also contained CCK-A receptor sites. High concentrations of CCK-A receptors were present in the substantia nigra zona compacta and also the ventral tegmental area and may be associated with dopamine cell bodies. Binding of 3H-MK-329 was also detected in parts of the caudate nucleus and ventral putamen. The detection, by autoradiographical means, of CCK-A receptors throughout the Cynomolgus monkey brain contrasts with similar studies performed using rodents and suggests differences in the density and, perhaps, the importance of CCK-A receptors in the primate as opposed to the rodent. The data suggest the possibility that CCK-A receptors may be involved in a number of important brain functions as diverse as the processing of sensory information from the gut, the regulation of hormone secretion, and the activity of dopamine cell activity.

There is evidence for the role of the cholecystokinin (CCK) neurotransmitter system in the neurobiology of panic disorder (PD). The CCK receptor agonist, CCK-tetrapeptide (CCK-4) fulfills criteria for a panicogenic agent and there is evidence that PD might be associated with an abnormal function of the CCK system. For example, PD patients show an enhanced sensitivity to CCK-4, and exhibit lower CSF and lymphocyte CCK concentration as compared to healthy controls (reviewed by Bradwejn et al.). Also, untreated PD patients display an increased CCK-4-induced intracellular Ca2+ mobilization in T cells relative to treated PD, depression and schizophrenia. The CCK receptors have been classified into two subtypes: CCK-A and CCK-B. We report here a study of polymorphisms in the CCK pre-pro hormone gene (CCK), CCK-AR, and CCK-BR in DSM-IV panic patients (n = 99) vs controls matched for gender and ethnicity. The CCK polymorphism revealed no association with PD. We identified a new polymorphism for the CCK-A receptor gene, and tested it in our sample, with negative results. A single nucleotide polymorphism has been found in the coding region of the CCK-B receptor gene (CCK-BR) and D Collier (personal communication) identified a highly polymorphic dinucleotide (CT)n microsatellite in the 5' regulatory region. For the CCK-B receptor gene polymorphism, PD patients showed a significant association. Our genetic dissection of the CCK system thus far suggests that the CCK-B receptor gene variation may contribute to the neurobiology of panic disorder.

BACKGROUND

Cholecystokinin (CCK)-receptor antagonists have been found to markedly reduce the severity of pancreatitis and improve survival in experimental animal models of acute pancreatitis. CCK appears to play an important role in the development and progression of acute pancreatitis, and the recent development of CCK antagonists has provided a new approach to the treatment of acute pancreatitis in humans.

OBJECTIVE

The therapeutic efficacy of a CCK-A receptor antagonist, loxiglumide, in patients with painful acute attacks of chronic pancreatitis was evaluated.

METHODS

A multicenter dose-response controlled trial was conducted at 110 institutions in Japan from June 1993 to December 1994. Chronic pancreatitis was diagnosed for all patients on the basis of the Japanese criteria for chronic pancreatitis. Two-hundred seven patients were randomized to oral treatment with loxiglumide (300, 600, and 1,200 mg/d) or placebo for 4 weeks. The efficacy of treatment was evaluated on the basis of clinical symptoms, physical signs, and serum pancreatic enzyme levels. The groups were comparable with respect to age, sex, etiology, complications, and previous treatment.

RESULTS

The improvement rate of the abdominal and/or back pain was 46% in the loxiglumide 300-mg group, 59% in the 600-mg group, and 52% in the 1,200-mg group, and it was 36% in the placebo group (600 mg versus placebo: p < 0.05). The physical signs evaluated--abdominal tenderness and resistance--improved in all three loxiglumide groups, and the serum pancreatic amylase and trypsin levels decreased significantly in the 600-mg group (p < 0.05). The overall clinical improvement rate was 46% in the 300-mg loxiglumide group, 58% in the 600-mg group, and 52% in the 1,200-mg group, and it was 34% in the placebo group.

CONCLUSIONS

These results indicate that oral administration of loxiglumide may be useful in the treatment of patients with acute, painful attacks of chronic pancreatitis, and 600 mg/d is recommended as a beneficial dosage.

We report here the cDNA cloning of a putative gastrin receptor from enterochromaffin-like (ECL) carcinoid tumor of Mastomys natalensis. For this study, we used the polymerase chain reaction technique to amplify transmembrane domain sequences related to rat pancreatic cholecystokinin (CCK)-A receptor from the ECL tumor cDNA library. The amino acid sequence deduced from the cloned cDNA showed 85.7% and 49.0% identity to canine parietal cell gastrin receptor and rat pancreatic CCK-A receptor, respectively. Ligand binding studies using COS-7 cells transfected with the cDNA showed the same binding specificity for gastrin and CCK-8 as the gastrin receptor on the Mastomys carcinoid tumor membrane. Both gastrin and CCK-8 elevated free cytosolic calcium concentration in COS-7 cells expressing the cloned receptor. RNA blot analysis revealed the expression of the gastrin receptor in both Mastomys stomach and brain.

Cholecystokinin (CCK) has been proposed to serve as a satiety signal in animals and humans. To further explore the role of CCK in humans, the effect on satiety and eating behavior of a specific CCK-receptor antagonist, loxiglumide, that preferentially inhibits peripheral (CCK-A) receptors was investigated. In a randomized, blind, four-period latin square design, 10 subjects received intravenous saline (placebo) or loxiglumide (10 mg/kg per hour) with concomitant intrajejunal perfusions of isotonic saline or fat (containing 50% corn oil and 3% albumin). Food intake and plasma CCK concentrations were assessed, and subjects scored their feelings of hunger and fullness in paired experiments. In placebo-treated subjects, the duration of the meal was shorter during fat perfusion (30 +/- 2 minutes vs. 35 +/- 2 minutes; P less than 0.01; mean +/- SEM). The amount of food intake was reduced (361 +/- 31 g vs. 454 +/- 35 g; P less than 0.05), and fluid ingestion was inhibited (490 +/- 31 mL vs. 625 +/- 38 mL; P less than 0.01). Loxiglumide did not affect any parameter and did not change the pattern of responses. In loxiglumide-treated subjects there was a 4-5-fold elevation in plasma CCK levels. These results confirm that jejunal infusion of lipid reduces the size of the meal and stimulates early satiety. The data imply that these effects are not mediated through peripheral endogenous CCK under these conditions.

OBJECTIVE

Supramaximal concentrations of cholecystokinin (CCK) or cerulein induce the intracellular activation of trypsinogen and the transcription factor NF-kappaB, a key regulator of inflammatory gene expression. Both events occur early in the development of an acute pancreatitis. The aim of this study was to examine the relationship between intracellular trypsinogen and NF-kappaB activation.

METHODS

We detected NF-kappaB-binding activity in electromobility shift assays, IkappaB proteolysis in Western analysis and endogenous IkappaB-kinase (IKKalpha and beta) activation using immune complex kinase assays following treatment with CCK in rat pancreatic lobules. To block intrapancreatic trypsinogen activation, a potent and cell-permeable serine-protease inhibitor, Pefabloc, was used.

RESULTS

CCK-induced IkappaBalpha degradation and subsequent NF-kappaB activation correlated closely with the catalytic activity of IKKs to phosphorylate IkappaBalpha in vitro. Activation is dose-dependent and peaked at 30 min. Doses of Pefabloc sufficient to inhibit trypsin activation reduced CCK-induced activation of NF-kappaB whereas TNF-alpha-induced NF-kappaB activation was not blocked but slightly increased. Moreover, treatment with Pefabloc as well as another serine protease inhibitor, FUT175, inhibited CCK-induced IKK activation.

CONCLUSIONS

These results suggest that intrapancreatic activation of trypsinogen may contribute to NF-kappaB signaling via IKK activation in cerulein pancreatitis. This also explains the fact that only doses of CCK which activate trypsinogen induce NF-kappaB activation in pancreatic acinar cells. Thus, trypsinogen activation is likely to modulate signaling events in acinar cells in the initial phase of acute pancreatitis.

We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.

Both cholecystokinin (CCK), a short-term meal-related satiety signal, and the ob protein leptin, a postulated long-term adiposity hormone, are thought to be important signals in the multiple interacting systems that control appetite and adiposity. We hypothesized that these hormones may synergistically interact to suppress feeding. Following IP administration of leptin (two doses of 50 micrograms each) and CCK (2, 4, 8, or 16 micrograms) total daily caloric intake was significantly reduced by leptin and CCK compared to leptin alone. These results support the hypothesis that CCK and leptin may synergistically interact to control long-term feeding.

The OLETF rat, lacking CCK-A receptors, provides an important model for identifying roles for CCK in the controls of food intake and body weight. OLETF rats are obese and diabetic and express deficits in the control of the size of individual meals. Meal size in OLETF rats is doubled and although meal number is decreased, the decrease is not sufficient to prevent hyperphagia. Analyses of patterns of hypothalamic gene expression in OLETF rats indicate the presence of a primary deficit in DMH NPY signaling. These data suggest an important role for CCK in controlling NPY expression in a population of non-leptin regulated hypothalamic neurons. In the absence of this control, NPY is overexpressed, contributing to hyperphagia and obesity. Thus, the obesity in the OLETF rats may be the outcome of two regulatory disruptions, one depending upon a peripheral within meal satiety pathway and the other depending upon a central pathway critical to overall energy balance.

OBJECTIVE

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

METHODS

TO at 10 mg/kg was orally administered, followed by 75 microg/kg CCK octapeptide injected subcutaneously three times after 1, 3 and 5 h. This whole procedure was repeated for 5 d. We determined the pancreatic weight/body weight ratio, the levels of pancreatic HSP60 and HSP72, and the secretion of pro-inflammatory cytokines. Repeated CCK octapeptide treatment resulted in typical laboratory and morphological changes of experimentally-induced pancreatitis.

RESULTS

TO significantly decreased the pancreatic weight/body weight ratio in CCK octapeptide-induced acute pancreatitis. TO also increased the pancreatic levels of HSP60 and HSP72. Additionally, the secretion of IL-6 and TNF-alpha decreased in the animals treated with TO.

CONCLUSIONS

TO may have a protective effect against CCK octapeptide-induced acute pancreatitis.

We have previously reported that adenovirus-mediated expression of preprocholecystokin (CCK) stimulates human and mouse islet cell proliferation. In follow-up studies, we became concerned that the CCK adenovirus might have been contaminated with a wild-type E1A-containing adenovirus. Here we show conclusively that the proliferative effects reported in the original paper in mouse and human islets were not due to CCK expression but rather to a contaminating E1A-expressing wild-type adenovirus. We also show, however, that CCK expression does have a proliferative effect in rat islets. We hope that our report of the steps taken to detect the wild-type virus contamination, and purification of the contributing viral stocks, will be helpful to other investigators, and that our experience will serve as a cautionary tale for use of adenovirus vectors, especially for studies on cellular replication.

The gastrointestinal peptide cholecystokinin (CCK) has been shown to stimulate growth of human pancreatic adenocarcinoma in vitro and in vivo, although CCK receptors have not been identified in pancreatic cancer cells. The purpose of this study was to characterize the CCK receptors in pancreatic cancer cells and to correlate the receptor binding studies with the trophic action of CCK agonists and antagonists. With the use of homogenates of PANC-1 human pancreatic cancer cell line grown in culture, the binding of 125I-labeled CCK octapeptide (125I-CCK-8) was examined under various conditions to characterize the CCK receptor. Specific and saturable binding of 125I-CCK-8 was detected in PANC-1 cells; data were consistent with a single binding site. Scatchard analysis yielded a binding affinity [dissociation constant (Kd)] of 2.8 nM and a binding capacity of 26 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, the presence of protease inhibitors, and pH and was sensitive to Na+, K+, Mg2+, and ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Competition experiments indicated that L-365,260, a selective CCK-B (gastrin) receptor antagonist, was the most potent displacer of 125I-CCK-8, and no significant displacement of binding was found with the selective CCK-A receptor antagonist. Growth of PANC-1 cells in culture was stimulated by CCK at a concentration consistent with the Kd, and CCK-stimulated growth was inhibited by the CCK-B receptor antagonist (L-365,260) not the CCK-A receptor antagonist (L-364,718).(ABSTRACT TRUNCATED AT 250 WORDS)

The actions of intravenous sulfated cholecystokinin octapeptide (CCK-8) on intraluminal pressure in the body of the stomach were studied in urethan-anesthetized rats. There was a dose-related decrease in pressure in response to CCK-8 over the range 0.3-33 pmol. Bilateral cervical vagotomy alone reduced the response to CCK-8 and together with splanchnic section abolished it. Hexamethonium also reduced the response. Vagotomy did not change the response to CCK-8 in hexamethonium-treated rats, but celiac ganglionectomy abolished it. Guanethidine and phentolamine, but not propranolol, significantly decreased the response to CCK-8; subsequent vagotomy abolished the response. Similarly, depletion of tissue catecholamines by pretreatment with 6-OH dopamine, reserpine, or celiac ganglionectomy together with vagal section abolished the effect of CCK-8. It concluded that CCK-8 decreases mean intragastric pressure in the rat by pathways involving both vagal and splanchnic nerves. The splanchnic pathway involves an alpha-adrenergic mechanism but is hexamethonium resistant. The vagal pathway is hexamethonium sensitive and nonadrenergic. Similar pathways may mediate the effect of CCK on gastric emptying.

The recent discovery that subsets of rat taste receptor cells (TRCs) express the peptide cholecystokinin (CCK) and that subsets of TRCs respond to CCK with altered potassium currents or elevated intracellular calcium via CCK-A receptor has lead to the hypothesis that CCK may play a novel signaling role within the taste bud, perhaps modifying tastant responses by co-transmission with a classic transmitter. To better understand this phenomenon, CCK-responsive TRCs were characterized for sensitivity to two bitter stimuli, quinine or caffeine, or to the neurotransmitter ACh using a ratiometric procedure with the calcium sensitive dye fura-2. In characterizing TRC responses to quinine, it was observed that quinine-induced elevations of intracellular calcium were not due to endogenous fluorescence of the quinine molecule. Most (60-70%) CCK-responsive cells were also sensitive to either bitter stimuli or to cholinergic stimulation. These data suggest that TRCs expressing CCK-receptors also express receptors to bitter stimuli and/or muscarinic receptors. They further support the notion of a putative modulatory role of CCK with convergence of multiple inputs occurring at the level of intracellular calcium.

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca(2+)-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca(2+)](i) decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK(A) receptor states. CCK stimulated an association of Lyn with PKC-delta, Shc, p125(FAK) and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130(CAS) or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125(FAK), PYK2, PKC-delta and Shc, which play central roles in CCK's effects on acinar cell function.

Nutrients in the intestine initiate changes in secretory and motor function of the gastrointestinal (GI) tract. The nature of the 'sensors' in the intestinal wall is not well characterized. Intestinal lipid stimulates the release of cholecystokinin (CCK) from mucosal entero-endocrine cells, and it is proposed that CCK activates CCK A receptors on vagal afferent nerve terminals. There is evidence that chylomicron components are involved in this lipid transduction pathway. The aim of the present study was to determine (1) the pathway mediating reflex inhibition of gastric motility and (2) activation of duodenal vagal afferents in response to chylomicrons. Mesenteric lymph was obtained from awake rats fitted with lymph fistulas during intestinal perfusion of lipid (Intralipid, 170 micromol h(-1), chylous lymph) or a dextrose and/or electrolyte solution (control lymph). Inhibition of gastric motility was measured manometrically in urethane-anaesthetized recipient rats in response to intra-arterial injection of lymph close to the upper GI tract. Chylous lymph was significantly more potent than control lymph in inhibiting gastric motility. Functional vagal deafferentation by perineural capsaicin or CCK A receptor antagonist (devazepide, 1 mg kg(-1), i.v.) significantly reduced chylous lymph-induced inhibition of gastric motility. The discharge of duodenal vagal afferent fibres was recorded from the dorsal abdominal vagus nerve in an in vitro preparation of the duodenum. Duodenal vagal afferent nerve fibre discharge was significantly increased by close-arterial injection of CCK (1-100 pmol) in 43 of 83 units tested. The discharge of 88% of CCK-responsive fibres was increased by close-arterial injection of chylous lymph; devazepide (100 microg, i.a.) abolished the afferent response to chylous lymph in 83% of these units. These data suggest that in the intestinal mucosa, chylomicrons or their products release endogenous CCK which activates CCK A receptors on vagal afferent nerve fibre terminals, which in turn initiate a vago-vagal reflex inhibition of gastric motor function.

Using gel, ion-exchange, and reverse-phase chromatography monitored by radioimmunoassays specific for five sequences of preprocholecystokinin (prepro-CCK), its processing products were measured in neutral and acid extracts of porcine cerebral cortex before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Three categories of peptides were found: biologically active peptides, i.e. peptides with the alpha-amidated COOH terminus Trp-Met-Asp-Phe-NH2, comprising large CCKs, i.e. peptides larger than CCK-58 and peptides eluting like CCK-58, CCK-33, and CCK-22; CCK-octapeptides in sulfated and traces of nonsulfated forms; and small CCKs, i.e. traces of CCK-7, large amounts of CCK-5, and modest concentrations of CCK-4 (the structures of CCK-5 and -4 were confirmed by sequence analysis); four NH2-terminal fragments, of which the two predominant ones correspond to the desnonapeptide fragments of CCK-58 and CCK-33; and COOH-terminal extended peptides corresponding to glycine-extended CCK-58, CCK-33, and CCK-8 in small but significant amounts. Thus, in addition to CCK-8 the porcine cerebral cortex synthesizes larger and smaller active CCK peptides in quantities of an order similar to those of CCK-8. The occurrence of these together with the NH2-terminal fragments and glycine-extended peptides can be explained only by the existence of different processing pathways for preproCCK. Consequently, the results suggest that cerebral CCK neurons are heterogeneous and comprise at least three populations with different biosynthetic machineries.

The potent and selective non-peptide cholecystokinin (CCK) antagonist L-364,718 (0.5-2.0 mg/kg s.c.) enhanced the analgesia induced by acute morphine treatment in the rat tail flick test. Chronic treatment with L-364,718 (1.0 mg/kg) prevented the development of tolerance to morphine analgesia (after a 6 day period of morphine treatment) but did not influence the onset of opioid dependence. Since L-364,718 is considerably more potent in inhibiting CCK binding to peripheral tissues than to brain membranes its interaction with morphine is surprising. The exact locus of this interaction, or whether it involves 'peripheral-type' (CCK-A) or 'central-type' (CCK-B) receptors is not known.

To identify the transduction mechanisms underlying gastric vagal afferent responses to gastric loads and cholecystokinin (CCK), we investigated the ability of specific CCK antagonists, acute pylorectomy, and cholinergic blockade to effect these vagal afferent responses. The CCK-B antagonist L-365,260 (10 pmol-1 nmol) failed to block the gastric vagal afferent response to gastric loads or 100 pmol CCK, while the CCK-A antagonist devazepide (100 pmol-100 nmol) competitively and dose dependently attenuated the response to CCK but not to gastric loads. Application of 100 nmol of the low-affinity CCK receptor antagonist CCK-JMV-180 also completely blocked the gastric vagal afferent response to 100 pmol CCK. Acute pylorectomy failed to block the gastric vagal afferent response to 100 pmol CCK or 2-ml gastric loads. Atropine sulfate administration (15 mg/rat) failed to block the gastric vagal afferent response to 100 pmol CCK or 2-ml gastric loads. These data suggest that 1) the vagal afferent response to CCK is mediated through CCK's interactions with vagal, rather than pyloric, CCK-A receptors, and 2) the vagal afferent responses to CCK and to gastric loads are mediated by dissociable, possibly independent, transduction mechanisms.

BACKGROUND

The cholecystokinin (CCK) family of peptides and receptors is present throughout the brain and gastrointestinal tract. The CCK receptors can be pharmacologically subdivided into two subtypes: CCK-A and CCK-B. CCK-A receptor is enriched in the pancreas of mice.

OBJECTIVE

To determine pancreatic functions in a CCK-A receptor deficient mouse mutant generated by gene targeting in embryonic stem cells. The targeting vector contained lacZ and neo insertions in exon 2.

METHODS

To examine exocrine functions, amylase release from the dispersed acini in vitro was examined. In the in vivo study, the mixture of bile-pancreatic juice was collected, and amylase, bicarbonate, and bile acid outputs were determined after the administration of various stimulants. The cystic duct of the gallbladder and the pylorus were ligated to exclude the involvement of gallbladder contraction and gastric acid. Pancreatic enzyme content was measured, and histologic examinations by HE and lacZ staining were conducted. To examine endocrine functions, oral glucose tolerance test (2 g/kg) was determined.

RESULTS

The body weight, pancreatic wet weight, and enzyme content in the pancreas were similar among the three genotypes. Amylase release in vivo and in vitro and bicarbonate secretion in vivo were not stimulated by CCK-8 in CCK-AR (-/-) mice, whereas the responses to other stimulants were substantial in (-/-) mice. Administration of secretin did not increase bicarbonate secretion regardless of genotype. A normal glucose tolerance was observed in (-/-) mice. Acinar cells, islets, and duct cells were stained by lacZ, and HE staining revealed no pathologic findings.

CONCLUSIONS

The CCK-A receptor is important for pancreatic exocrine secretion, but not essential for maintaining glucose concentration and pancreatic growth in mice.

Expressions of the CCK-A and B receptor genes in fetal and adult pancreas of OLETF rats were examined by the reverse transcriptase polymerase chain reaction followed by Southern blot hybridization. The pancreatic responses to various stimulants were examined in vitro and results were compared with those of control (LETO) rats. CCK-A receptor mRNA was not expressed in the fetal pancreas of either strain or in the adult pancreas of OLETF rats, but was expressed in the adult pancreas of LETO rats. CCK-B receptor mRNA was expressed in fetal and adult pancreas in both strains. Southern blot hybridization indicated a difference in gene structure in the two strains. The maximal effective concentrations of neuromedin C, carbachol, and secretin for amylase secretion and intracellular Ca2+ movement stimulated by carbachol and neuromedin C were similar in the two strains. CCK-8 and the non-sulfated form stimulated amylase secretion only in LETO rats. These results suggest that OLETF rats are a new model of a congenital defect of the CCK-A receptor gene and should be useful for determining CCK receptor function.