Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons.

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.

Related papers:

title: Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin.
abstract: Poly(A) RNA was isolated from a rat medullary thyroid carcinoma that exhibited high levels of immunoreactive cholecystokinin (CCK). Double-stranded cDNA was synthesized from the poly(A) RNA and inserted into the Pst I site of pBR322. Bacterial colonies containing CCK cDNA were identified using the hybridization probe d(T-C-C-A-T-C-C-A-N-C-C-C-A-T-G-T-A-G-T-C). The sequence of the probe was deduced from the known amino acid sequence of porcine CCK-8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2. The nucleotide sequence of the cDNA complementary to the mRNA of rat preprocholecystokinin was determined. The cDNA contains 33 nucleotides in the 5'-noncoding region, 199 nucleotides in the 3'-noncoding region, and 345 nucleotides coding for a precursor to CCK, which is 115 amino acids (Mr, 12,826). Examination of the rat CCK gene revealed a suggested transcriptional control sequence analogous to the "TATA" sequence located 33 nucleotides upstream from a proposed transcriptional start site. The amino acid sequence of CCK-39 is flanked by both amino-terminal and carboxyl-terminal extensions. Analysis of CCK mRNA showed that it is approximately equal to 750 nucleotides long. CCK mRNA of the rat brain and intestine appeared to be identical in size to the CCK mRNA of the carcinoma.

title: Characterization of the major form of cholecystokinin in human intestine: CCK-58.
abstract: Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

title: Cholecystokinin cells.
abstract: Cholecystokinin (CCK) is an important hormonal regulator of the digestive process. CCK cells are concentrated in the proximal small intestine, and hormone is secreted into the blood upon the ingestion of food. The physiological actions of CCK include stimulation of pancreatic secretion and gallbladder contraction, regulation of gastric emptying, and induction of satiety. Therefore, in a highly coordinated manner, CCK regulates the ingestion, digestion, and absorption of nutrients. CCK is produced by two separate cell types: endocrine cells of the small intestine and various neurons in the gastrointestinal tract and central nervous system. Accordingly, CCK can function as either a hormone or a neuropeptide. This review focuses on the physiology of the CCK cell in the intestine and, in particular, on how the CCK cell is regulated to secrete its hormone product. The effects of ingested nutrients on the CCK cell and the intracellular messenger systems involved in controlling secretion are reviewed. A summary is provided of recent studies examining the electrophysiological properties of CCK cells and newly discovered proteins that act as releasing factors for CCK, which mediate feedback pathways critical for regulated secretion in the intact organism.

title: High-affinity CCK-A receptors on the vagus nerve mediate CCK-stimulated pancreatic secretion in rats.
abstract: Cholecystokinin (CCK) receptors are found on vagal afferent fibers. In pancreatic acini, CCK receptors exist in high- and low-affinity states. The aim of this study was to identify the vagal CCK-A receptor affinity state that mediates the effect of CCK on pancreatic protein secretion. Using a rat model with a pancreatic-biliary cannula, we studied the effects of CCK-JMV-180 on exocrine pancreatic function. CCK-JMV-180 acts as an agonist on high-affinity CCK receptors and as an antagonist on low-affinity CCK receptors. Infusion of CCK-JMV-180 (22-88 micrograms.kg-1.h-1) caused dose-dependent increases in pancreatic protein secretion, which were blocked by the CCK-A receptor antagonist L-364,718. Acute vagotomy in anesthetized rats and perivagal application of capsaicin in conscious rats abolished pancreatic responses to CCK-JMV-180 at 22 and 44 micrograms.kg-1.h-1. CCK-JMV-180 did not reduce pancreatic responses to CCK octapeptide infusion at 20 and 40 pmol.kg-1.h-1. To demonstrate that endogenously released CCK also acts on high-affinity CCK-A receptors, we showed that in conscious rats intraduodenal infusion of 18% casein produced a threefold increase in protein secretion and elevated plasma CCK levels from 0.7 to 8.4 pM. Infusion of CCK-JMV-180 at 44 micrograms.kg-1.h-1 failed to inhibit pancreatic responses to casein. In separate studies, perivagal application of 1% capsaicin inhibited 95% and 90% of the pancreatic responses to casein and casein combined with intravenous CCK-JMV-180, respectively. The neurotoxic effect of capsaicin on small-diameter sensory vagal fibers was verified by immunohistochemical and retrograde tracing studies. In conclusion, we demonstrated that in contrast to their effect on satiety, which is mediated by vagal low-affinity CCK-A receptors, exogenous CCK and endogenous CCK under physiological conditions act through high-affinity CCK-A receptors to mediate pancreatic protein secretion. These findings suggest that different affinity states of the vagal CCK receptors mediate different digestive functions.

title: Cholecystokinin inhibits gastric emptying and contracts the pyloric sphincter in rats by interacting with low affinity CCK receptor sites
abstract: The aim of these experiments was to characterize the receptor affinity state through which CCK produces pyloric contraction and inhibits gastric emptying in the rat using the novel CCK heptapeptide analog CCK-JMV-180. CCK-JMV-180 has been demonstrated to act as a functional agonist at high affinity pancreatic CCKA receptors but as a functional antagonist at CCKA low affinity receptors. CCK-8 (1, 3.2 and 10 nM) induced dose dependent tension increases in isolated pyloric segments. CCK-JMV-180 (3.2 microM) or vehicle failed to mimic this action when administered alone but blocked the ability of CCK-8 (3.2 nM) to induce tension increases. CCK-8 (2 micrograms/kg) also inhibited the gastric emptying of physiological saline. CCK-JMV-180 (320 and 1000 micrograms/kg) failed to inhibit emptying when administered alone but dose dependently antagonized CCK induced inhibition of gastric emptying. Thus, in both preparations CCK-JMV-180 acted as a functional CCK antagonist. This profile is consistent with the interpretation that the actions of CCK in pyloric contraction and the inhibition of gastric emptying are mediated through CCK's interactions with receptors functionally similar to pancreatic low affinity sites.

title: Cholecystokinin inhibits gastric motility and emptying via a capsaicin-sensitive vagal pathway in rats.
abstract: The pathway by which cholecystokinin octapeptide (CCK-8) inhibits motility of the proximal stomach and the role of this pathway in the CCK-induced delay in gastric emptying of a liquid meal has been studied in rats by selective destruction of vagal afferent C-fibers using bilateral perineural application of the sensory neurotoxin, capsaicin, 3 or 4 days prior to the experiment. The capsaicin treatment significantly attenuated the decrease in intragastric pressure in urethan-anesthetized rats in response to CCK-8 (0.1-100 pmol iv) compared with vehicle-treated controls. Removal of the celiac-superior mesenteric ganglion completely abolished the inhibitory action of CCK-8 on gastric motility in these rats. In contrast, only celiac ganglionectomy in combination with vagotomy abolished the CCK-8 effect in vehicle-treated controls. Intravenous injection of CCK-8 (300 pmol) 5 min before intragastric administration of a methylcellulose solution decreased gastric emptying by 55% in conscious control or vehicle-treated rats. Perivagal capsaicin treatment abolished the delay in gastric emptying induced by CCK-8. In addition, capsaicin treatment alone significantly increased gastric emptying. These results demonstrate that CCK-8 decreases gastric motility in the gastric corpus and delays gastric emptying by a capsaicin-sensitive vagal afferent pathway. These same afferent fibers may also play a physiological role in the gastric emptying of liquids.

title: Capsaicin pretreatment attenuates suppression of food intake by cholecystokinin.
abstract: Capsaicin, the pungent principal in red pepper, has been shown to damage small-diameter peptide-containing sensory neurons. Suppression of feeding by cholecystokinin octapeptide (CCK OP) was attenuated after systemic pretreatment with capsaicin. Capsaicin pretreatment did not attenuate suppression of food intake by intragastric preloads. Pretreatment of rats with microgram quantities of capsaicin injected into the fourth ventricle, near the sites of vagal afferent termination, also attenuated CCK OP-induced suppression of food intake. These results suggest that the satiety-inducing effects of CCK OP are mediated, at least in part, by capsaicin-sensitive afferent neurons.

title: Afferent axons in abdominal vagus mediate satiety effect of cholecystokinin in rats.
abstract: Selective section of afferent vagal axons that reach below the diaphragm blocks the satiating effect of peripherally administered cholecystokinin in the rat. Section of the analogous efferent axons has no effect. After the behavioral tests, the selective axonal sections were confirmed with horseradish peroxidase histochemistry.

title: Differential effects of CCK-JMV-180 on food intake in rats and mice
abstract: Boc-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2-phenylether ester (CCK-JMV-180) has been reported to be a CCK-based heptipeptide with novel in vitro properties. Based on studies conducted in rat and mouse pancreatic acini, it has been proposed that the compound acts as an agonist at the high-affinity site and an antagonist at the low-affinity site in the rat, but as an agonist at both sites in the mouse. In the present study, we examined the effects of CCK-JMV-180 on locomotor activity in the rat and on intake of a liquid diet in the rat and mouse. Although CCK-JMV-180 slightly reduced activity on its own in the rat, it completely reversed the suppression produced by coadministration of CCK-8. In rat feeding studies, CCK-JMV-180 failed to suppress intakes of a liquid diet, but was able to antagonize the anorectic effects of CCK-8. In contrast, in the mouse CCK-JMV-180 potently suppressed intakes on its own, and this effect was blocked by pretreatment with the selective CCK-A receptor antagonist, A-70104. The results of these studies suggest that similar receptor mechanisms are involved in CCK's ability to inhibit food intake in vivo and its effects on pancreatic function in vitro.

title: Type A CCK receptors mediate satiety effects of intestinal nutrients
abstract: Previous work indicates that endogenous CCK mediates suppression of sham feeding by some intraintestinal nutrients. To test whether the mechanism involved is dependent upon action at type A or type B CCK receptors, we examined the ability of CCKA (devazepide) and CCKB (L-365,260) receptor antagonists to attenuate the suppression of sham feeding by intraintestinal oleic acid, maltotriose, or L-phenylalanine. Suppression by oleic acid or maltotriose was dose dependently attenuated by intraperitoneal administration of the CCKA receptor antagonist, as was suppression by exogenous CCK. The CCKB receptor antagonist failed to attenuate the suppression of sham feeding by these nutrients. Neither receptor antagonist attenuated the suppression of sham feeding induced by intraintestinal L-phenylalanine. These results suggest that suppression of sham feeding by intestinally infused oleic acid and maltotriose is mediated by endogenous CCK acting at CCKA receptors.

title: CCK satiety is differentially mediated by high- and low-affinity CCK receptors in mice and rats.
abstract: Cholecystokinin-JMV-180 (JMV-180) is an analogue of cholecystokinin C-terminal octapeptide (CCK-8), which has been shown to be an agonist at the proposed CCK pancreatic high-affinity site and a functional antagonist at the pancreatic low-affinity site in rats and to have agonist activity at both high- and low-affinity sites in the mouse. In this study we used JMV-180 to evaluate the potential participation of these two CCK-A sites in the satiety effect of CCK-8 in rats and mice. When tested at doses that ranged from 0.01 to 9.2 mumol/kg, JMV-180 did not reliably affect food intake of solid or liquid test diets in rats. When combined with CCK-8 (3.2 or 8.5 nmol/kg) JMV-180 dose dependently reversed the satiety effect of CCK-8. In contrast to these results in the rat, both JMV-180 (3.7-14.8 mumol/kg) and CCK-8 (1.7-6.8 nmol/kg) dose dependently reduced the intake of 20% sucrose in mice. Both CCK-8- and JMV-180-induced suppression of food intake were attenuated by the CCK-A antagonist MK-329 (24.8 nmol/kg). The results of these studies suggest that agonist activity at sites pharmacologically similar to the CCK pancreatic high-affinity site is not sufficient for expression of CCK satiety, whereas agonist activity at low-affinity-like sites is necessary to reduce food intake. Thus the anorexic activity of CCK appears to be mediated through an interaction with a receptor site pharmacologically similar to the pancreatic low-affinity CCK receptor site.

title: CCK-receptor antagonists attenuate suppression of sham feeding by intestinal nutrients.
abstract: To test the possibility that endogenous cholecystokinin (CCK) participates in suppression of sham feeding by intraintestinal nutrient infusions, we examined the effect of CCK-receptor antagonists on the suppression of sham feeding by intraintestinally infused oleic acid, maltose or L-phenylalanine (L-Phe). In addition, we monitored amylase activity in the intestinal lumen during some sham feeding experiments and measured plasma CCK in parallel experiments using intestinally infused animals that were not feeding. Suppression of sham feeding by oleic acid or maltose was attenuated by CCK-receptor antagonists, while suppression of sham feeding by L-Phe was not. Oleate infusion increased plasma CCK concentration and luminal amylase activity. Oleate-induced increase in luminal amylase activity was attenuated by a CCK-receptor antagonist. Intraintestinal maltose or L-Phe did not increase plasma CCK concentration or luminal amylase activity, suggesting that they did not release endocrine CCK. These results suggest 1) that endogenous CCK mediates suppression of sham feeding by oleate and maltose but not by L-Phe and 2) that CCK participating in suppression of feeding by intestinal stimuli might not be of endocrine origin.

title: Suppression of sham feeding by intraintestinal nutrients is not correlated with plasma cholecystokinin elevation.
abstract: To test the possibility that blood-borne cholecystokinin (CCK) participates in suppression of sham feeding by intestinal nutrients in rats, we examined the ability of oleate, maltose, L-phenylalanine (L-Phe), and casein to suppress sham ingestion of 15% sucrose solution. Plasma CCK concentrations were measured in parallel experiments in which the same intestinal nutrients were infused in rats that were not feeding. Intraintestinal oleate suppressed sham feeding and elevated plasma CCK concentrations. Casein infusion produced plasma CCK concentrations similar to those produced by oleate but did not suppress sham feeding. Both maltose and L-Phe suppressed sham feeding but failed to significantly elevate plasma CCK. Previously we demonstrated that CCK receptor antagonists attenuate suppression of sham feeding by intestinal infusion of either oleate or maltose, suggesting that endogenous CCK participates in suppression of sham feeding by some intestinal nutrients. The results of the study reported here suggest that plasma CCK levels after nutrient infusion are not correlated with suppression of sham feeding. Therefore, the mechanism of CCK's participation in nutrient-induced suppression of sham feeding may not depend on stimulus-induced elevation of plasma CCK.

title: Gastric endocrine cells: gene expression, processing, and targeting of active products.
abstract: Endocrine cells of the gastric epithelium secrete biologically active peptides and small messenger molecules such as histamine, serotonin, and gamma-aminobutyric acid. The secretory products may act locally (paracrine or autocrine effects) or at distant targets after delivery in the circulation (hormonal effects). the contents of the gastric lumen control both secretion of gastric endocrine cells and the expression of genes involved in the synthesis of their active secretory products; in some cases, gene regulation may occur over periods as short as that required for digestion of a single meal. The conversion of inactive peptide precursors to their active forms takes place during transit along the secretory pathway and is only completed after sequestration in secretory granules. the processing of the gastrin precursor provides a useful model for studying prohormone processing. Generation of the well-known amidated gastrins requires prohormone cleavage and COOH-terminal amidation; the products stimulate acid secretion and mucosal growth. However, recent work indicates that biosynthetic intermediates that do not stimulate acid secretion may nevertheless act at a novel receptor to stimulate growth, so that control of prohormone processing determines which of two alternative types of biologically active peptide is released by gastrin cells. Gastric endocrine cells also have the capacity to accumulate small messenger molecules in secretory vesicles, via proton exchangers. Recent work indicates physiological regulation of the expression of genes encoding cytosolic enzymes such as histidine decarboxylase, which converts histidine to histamine, and of secretory granule transporters such as vesicular monoamine transporter type 2, which concentrates amines in secretory vesicles. Together these findings suggest that modulation of regulatory peptide and amine biosynthesis in gastric endocrine cells constitutes a primary response of the stomach to the arrival of a meal.

title: Infusion of CCK-8 into hepatic-portal vein fails to reduce food intake in rats.
abstract: If the putative satiating effect of endogenous cholecystokinin (CCK) is produced through a circulating hormonal mechanism, then administration of exogenous CCK into the hepatic-portal vein should decrease meal size. To test this, one form of endogenous CCK, the C-terminal octapeptide CCK-8, was infused intraportally in doses of 4 and 8 micrograms/kg just prior to a test meal. Neither dose decreased food intake after intraportal infusion even though intraperitoneal administration of 4 micrograms/kg CCK-8 decreased meal size approximately 50% in the same rats. The results suggest that if endogenous CCK-8 has a satiating effect, it acts primarily through a paracrine mechanism.

title: CCK Elicits and Modulates Vagal Afferent Activity Arising from Gastric and Duodenal Sites
abstract: We have begun to identify and characterize the locations and response profiles of vagal afferent fibers sensitive to CCK in the rat upper gastrointestinal tract. We found gastric and duodenal vagal afferent fibers that respond to CCK and to intraluminal loads. CCK both sensitizes and amplifies the response to loads in these fibers but may do so through separate transduction mechanisms. Thus, meal-related signals arising from the presence of gastroduodenal loads and the release of endogenous CCK can be integrated at the level of the peripheral afferent vagus nerve. These findings are consistent with behavioral results, demonstrating that combinations of gastric loads and exogenous CCK are more effective in suppressing food intake than is either stimulus presented alone. Our findings that both gastric and duodenal vagal afferent fibers are sensitive to CCK suggest that meal-related CCK may act at a range of peripheral neural sites linking the upper gastrointestinal tract to the central nervous system substrates underlying the control of food intake. The mode of activation of gastric vagal afferent by endogenously released CCK may be an endocrine action of intestinally derived CCK. Alternatively, the novel finding of duodenal load-sensitive vagal afferents close to a site of CCK release provides a potential for local paracrine actions of endogenous CCK in the mediation of satiety.

title: Effects of cholecystokinin (CCK-8) on two classes of gastroduodenal vagal afferent fibre.
abstract: In order to investigate the vagal afferent pathway responsible for the previously reported effects of cholecystokinin (CCK) on gastric emptying and food intake, single afferent fibres were recorded from the cervical vagus of urethane-anaesthetized ferrets. Sixty tension receptor afferents with receptive fields in the corpus, antrum, duodenum, jejunum and ileum all showed a resting level of discharge which was augmented powerfully by distension of the segment containing the ending. Close intraarterial injection of CCK-8 (100-200 pmol) caused relaxation in proximal regions, but enhanced contractile activity in more distal regions. Mechanoreceptor discharge closely followed intraluminal pressure at all times, indicating a sensitivity primarily to tension and no direct sensitivity to CCK. Only duodenal tension receptors were significantly excited by CCK (due to increased contractile activity), whereas those in the stomach showed a net decrease. Thirty-seven mucosal receptors from the corpus, antrum, duodenum and jejunum showed responses to luminal stimuli: predominantly light stroking, acidity and hypertonicity as has been previously described. No responses to glucose or amino-acid infusions could be evoked. However, mucosal fibres showed a strong sensitivity to close-intraarterially injected CCK-8 (3-200 pmol) in 19/26 fibres tested. These responses were unaffected by cholinergic blockade when tested. The data strongly suggest that in the ferret only vagal mucosal receptors are directly sensitive to CCK-8. These fibres are therefore likely candidates for mediating some of the reflex and behavioural effects of CCK when it is released from the gastrointestinal tract and acts directly on vagal sensory endings.

title: CCK-8 activates hepatic vagal C-fiber afferents
abstract: Intravenous administration of 2 micrograms/kg CCK-8 increased the single unit activity of 54% of hepatic vagal afferent fibers. Conduction velocity tests indicated that all of these units were C fibers. The increase in hepatic vagal activity produced by CCK-8 was significantly reduced by i.v. administration of 200 micrograms/kg of the CCKA receptor antagonist devazepide. Control comparisons indicated that this reduction was not an artifact of tachyphylaxis resulting from repeated administration of CCK-8. Further, the inability of pretreatment with atropine and hexamethonium to reduce the increases in hepatic vagal activity produced by CCK-8 suggests that the latter effect was not secondary to changes in gastrointestinal motor function. These outcomes demonstrate that activation of CCKA receptors by CCK-8 increases hepatic vagal afferent activity and support the view that the duodenal satiety action of CCK is mediated by the hepatic branch.

title: Mechanical properties and sensitivity to CCK of vagal gastric slowly adapting mechanoreceptors.
abstract: Sixty single afferent fibers with endings in the stomach wall were isolated from the cervical vagus of urethan-anesthetized Sprague-Dawley rats. All the fibers, most of which were spontaneously active, increased their discharge after gastric distension or during spontaneous contractions of the stomach. Because of this and the characteristic dynamic and static features of their response to inflation and deflation, they were identified as in-series tension receptors. Certain features of their responses, previously suspected from studies on reflex modulation of vagal efferent fibers or brain stem neurons, were directly confirmed. These included a broad range of mechanical thresholds and spontaneous firing frequencies that were correlated and a sensitivity only to dynamic stretch and active contraction in the highest threshold endings. The tension receptors could also be activated by circulating cholecystokinin, an effect unrelated to changes in intraluminal pressure and hence gastric wall tension, suggesting that there may be humoral modulation of visceral sensory signals that might be relevant to several behavioral situations, such as food intake regulation.

title: Diminished satiation in rats exposed to elevated levels of endogenous or exogenous cholecystokinin.
abstract: Rats maintained on a high-fat (HF) diet exhibit reduced sensitivity to the satiation-producing effect of exogenous CCK. Because more CCK is released in response to HF meals than low-fat (LF) meals, we hypothesized that increased circulating CCK associated with ingestion of HF diets contributes to the development of decreased CCK sensitivity. To test this hypothesis, we implanted osmotic minipumps filled with either NaCl or CCK octapeptide into the peritoneal cavity. Subsequently, we examined the effect of intraperitoneal NaCl or CCK (0.5 microg/kg) injection on 30-min food intake. CCK significantly reduced 30-min food intake less in rats implanted with CCK-releasing minipumps compared with those with NaCl-releasing minipumps. Because dietary protein is a potent releaser of endogenous CCK, we hypothesized that rats adapted to a high-protein (HP) diet might also exhibit reduced sensitivity to exogenous CCK. Therefore, in a second experiment, we examined CCK-induced reduction of food intake in rats maintained on LF and rats maintained on HF or HP. Ingestion of LF stimulates very little endogenous CCK secretion, whereas both HF and HP markedly increase plasma CCK concentrations. Both doses of CCK reduced food intake significantly less in HF and HP rats compared with LF rats. There were no differences in 24-h food intake, body weight, or body fat composition among LF-, HF-, and HP-fed rats. These results are consistent with the hypothesis that sustained elevation of CCK either by infusion of exogenous CCK or by dietary-induced elevation of plasma CCK contributes to the development of reduced sensitivity to exogenous CCK.

title: Rats maintained on high-fat diets exhibit reduced satiety in response to CCK and bombesin
abstract: Rats maintained on high-fat diets often exhibit increased food intake and weight gain. We hypothesized that high-fat diets might result in reduced sensitivity to hormonal signals responsible for terminating food intake--satiety signals. The intestinal hormone cholecystokinin (CCK) and the gastrointestinal neuropeptide, bombesin (BBS) both have been proposed as satiety signals. To determine whether maintenance on high-fat diets alters sensitivity to satiating effects of CCK and bombesin (BBS), rats were maintained on a low fat diet (LF), a high-fat diet that was isocaloric with the low-fat diet (HF), or one of two hypercaloric high-fat diets (HF-1, HF-2) that differed from HF and LF in fat, fiber, and total caloric content. CCK and bombesin reduced food intake significantly less in rats maintained on high-fat diets, compared to those on the low fat diet. Neither high caloric intake, which was associated with increased body weight gain on the two hypercaloric diets, nor fiber content of the diet accounted for the reduced response of HF rats to CCK. Rather, reduced sensitivity to CCK was related only to the high proportion of calories taken as fat. We also determined whether reduced CCK sensitivity was due to the maintenance on a particular diet or to the diet eaten during a CCK test. After CCK, rats maintained on LF reduced food intake more (49%) than rats maintained on HF (22%), regardless of whether they ate HF or LF during the CCK test itself. These findings indicate that maintenance of rats on high-fat diets reduces sensitivity to some peptide satiety signals. Reduced sensitivity to satiety signals might contribute to overeating and obesity often observed when rats are maintained on high-fat diets.

title: Vagotomy decreases excitability in primary vagal afferent somata.
abstract: Standard patch-clamp and intracellular recording techniques were used to monitor membrane excitability changes in adult inferior vagal ganglion neurons (nodose ganglion neurons, NGNs) 5 days following section of the vagus nerve (vagotomy). NGNs were maintained in vivo for 5 days following vagotomy, and then in vitro for 2-9 h prior to recording. Vagotomy increased action potential (AP) threshold by over 200% (264 +/- 19 pA, mean +/- SE, n = 66) compared with control values (81 +/- 20 pA, n = 68; P < 0.001). The number of APs evoked by a 3 times threshold 750-ms depolarizing current decreased by >70% (from 8.3 to 2.3 APs, P < 0.001) and the number of APs evoked by a standardized series of (0.1-0.9 nA, 750 ms) depolarizing current steps decreased by over 80% (from 16.9 APs to 2.6 APs, P < 0.001) in vagotomized NGNs. Similar decreases in excitability were observed in vagotomized NGNs in intact ganglia in vitro studied with "sharp" microelectrode techniques. Baseline electrophysiological properties and changes following vagotomy were similar in right and left NGNs. A "sham" vagotomy procedure had no effect on NGN properties at 5 days, indicating that changes were due to severing the vagus nerve itself, not surrounding tissue damage. NGNs isolated after being maintained 17 h in vivo following vagotomy revealed no differences in excitability, suggesting that vagotomy-induced changes occur some time from 1-5 days after injury. Decreased excitability was still observed in NGNs isolated after 20-21 days in vivo following vagotomy. These data indicate that, in contrast to many primary sensory neurons that are thought to become hyperexcitable following section of their axons, NGNs undergo a marked decrease in electrical excitability following vagotomy.

title: Characterization of CCK(A) receptor affinity states and Ca(2+) signal transduction in vagal nodose ganglia.
abstract: CCK(A) receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCK(A) receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca(2+) transient followed by a sustained Ca(2+) plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca(2+) oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCK(A) receptors, primarily elicited Ca(2+) oscillations (29% of cells responded). CCK-OPE inhibited the Ca(2+) transient induced by 1 nM CCK-8 but not by carbachol and high K(+). This result suggests the presence of high- and low-affinity states of CCK(A) receptors on nodose ganglia. We further demonstrated that nicardipine (10 microM) but not omega-conotoxins GVIA and MVIIC (10-100 nM) abolished Ca(2+) signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca(2+) channel and not an N- or Q-type Ca(2+) channel is coupled to CCK(A) receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca(2+) transient and inhibited CCK-8-evoked Ca(2+) signaling, indicative of G protein(s) involvement in CCK(A) receptor activity. The G(q) protein antagonist Gp antagonist-2A (10 microM) also abolished the action of CCK-8. This study indicates that CCK(A) receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCK(A) receptors elicits Ca(2+) oscillations, whereas stimulation of low-affinity CCK(A) receptors evokes a sustained Ca(2+) plateau. These Ca(2+)-signaling modes are mediated through the L-type Ca(2+) channel and involve the participation of G(q) protein.

title: CCK-B receptor: chemistry, molecular biology, biochemistry and pharmacology
abstract: Cholecystokinin (CCK) is a peptide originally discovered in the gastrointestinal tract but also found in high density in the mammalian brain. The C-terminal sulphated octapeptide fragment of cholecystokinin (CCK8) constitutes one of the major neuropeptides in the brain; CCK8 has been shown to be involved in numerous physiological functions such as feeding behavior, central respiratory control and cardiovascular tonus, vigilance states, memory processes, nociception, emotional and motivational responses. CCK8 interacts with nanomolar affinities with two different receptors designated CCK-A and CCK-B. The functional role of CCK and its binding sites in the brain and periphery has been investigated thanks to the development of potent and selective CCK receptor antagonists and agonists. In this review, the strategies followed to design these probes, and their use to study the anatomy of CCK pathways, the neurochemical and pharmacological properties of this peptide and the clinical perspectives offered by manipulation of the CCK system will be reported. The physiological and pathological implication of CCK-B receptor will be confirmed in CCK-B receptor deficient mice obtained by gene targeting (Nagata el al., 1996. ). Moreover, CCK receptor gene structure, deletion and mutagenesis experiments, and signal transduction mechanisms will be discussed.

title: CCK receptor antagonists.
abstract: 1. The peptide hormone and neurotransmitter, cholecystokinin, is widely distributed throughout the gastrointestinal tract and central nervous system and mediates a diverse number of biological functions. 2. Two receptor subtypes, CCK-A and CCK-B, have been identified by both pharmacological characterization and molecular cloning. The CCK-A receptor is the predominant peripheral CCK receptor subtype and the CCK-B receptor is the predominant central CCK receptor. In addition, there are discrete populations of CCK-A receptors in the brain and CCK-B receptors are present in gastric mucosa. 3. Subtype selective antagonists have been developed which discriminate between the two receptor subtypes. One of the major chemical classes has exploited a benzodiazepine template present in asperlicin which was initially discovered in a natural product screen for CCK receptor antagonists. 4. The structurally related benzodiazepines L-365,260, L-740,093, and YM022 are selective antagonists of the CCK-B receptor subtype. Their in vitro pharmacological profiles have been characterized using the human CCK-B receptor expressed in CHO cells. 5. L-365,260 behaves in a manner consistent with that of a competitive antagonist and both L-740,093 and YM022 behave as insurmountable CCK-B receptor antagonists in vitro.

title: Structure-antigastrin activity relationships of new (R)-4-benzamido-5-oxopentanoic acid derivatives.
abstract: New (R)-4-benzamido-5-oxopentanoic acid derivatives were synthesized by a stereoconservative procedure and evaluated in vitro for their capacity to inhibit the binding of [125I](BH)-CCK-8 to either rat peripheral (CCK-A) or central (CCK-B) CCK receptors, or the binding of [3H]pentagastrin to rabbit gastric glands, as well as to inhibit, in vivo, the acid secretion induced by pentagastrin infusion in the perfused rat stomach. The parent compound of this series (lorglumide) is the first nonpeptidic, potent and selective antagonist of the CCK-A receptor. Chemical manipulations of the structure of lorglumide led to the discovery of selective antagonists of the CCK-B/gastrin receptors. Structure-activity relationships are discussed. Some of these new derivatives exhibit different affinities with rabbit gastric gland cells and rat cortex membranes, suggesting that the stomach gastrin receptor (arbitrarily termed CCK-B1 receptor) is not as closely related to the CCK central receptor (termed CCK-B2) as previously hypothesized. The antigastric activity of the most potent compound of the series, i.e. (R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan- 8-yl)-5-oxopentanoic acid (compound 28, CR 2194) was further evaluated in vivo: in the first hour after administration the compound inhibits acid secretion induced by pentagastrin infusion, in both cat and dog (in the cat with gastric fistula and in the dog with Heidenhain pouch), with ID50s (mg/kg) of 15.5 (iv) (cat), 8.7 (IV) (dog) and 24.2 (oral) (Heidenhain dog). The characteristics of CR 2194, that is, the selectivity for the gastrin receptor, the simple nonpeptidic molecular structure, and the activity after oral administration, indicate that this compound is a useful tool in the study of the biological effects of gastrin and a potential agent for diagnostic or therapeutic use.

title: CCK-JMV-180: a peptide that distinguishes high-affinity cholecystokinin receptors from low-affinity cholecystokinin receptors.
abstract: When pancreatic acini are incubated with increasing concentrations of cholecystokinin octapeptide (CCK-8) the dose-response curve for stimulation of enzyme secretion increases, reaches a maximum and then decreases. The upstroke of the dose-response curve reflects occupation of high-affinity, stimulatory CCK receptors (Kd 69 pM), whereas the downstroke of the dose-response curve reflects occupation of low-affinity inhibitory CCK receptors (Kd 10 nM). In the present study, we used dispersed acini from rat pancreas to examine the effects of CCK-JMV-180, an analogue of the C-terminal heptapeptide of CCK (CCK-7) having the structure BOC-Tyr(SO3) Ahx-Gly-Trp-Ahx-Asp2 phenylethyl ester. CCK-JMV-180 inhibited binding of 125I-labelled CCK-8 to pancreatic acini. Computer analysis of the dose-inhibition curve indicated that CCK-JMV-180 interacted with both classes of CCK receptor and had a Kd of 2.2 nM for high-affinity CCK receptors and a Kd of 19 nM for low-affinity CCK receptors. Occupation of high-affinity CCK receptors by CCK-JMV-180 caused a 14-fold increase in amylase secretion, the same increase caused by occupation of these high-affinity receptors by CCK-7 or CCK-8. Occupation of low-affinity CCK receptors by CCK-JMV-180 did not alter amylase secretion, in contrast to occupation of these low-affinity receptors by CCK-7 or CCK-8, each of which caused inhibition of amylase secretion. Furthermore, occupation of the low-affinity CCK receptors by CCK-JMV-180 reversed the inhibition of amylase secretion caused by a supramaximal concentration of CCK-8. The present results indicate that CCK-JMV-180 interacts with high-affinity CCK receptors and with low-affinity CCK receptors, and has a functionally distinct action at each class of receptor: CCK-JMV-180 is an agonist at the high-affinity receptors and a competitive antagonist at the low-affinity receptors.

title: CCK antagonists reveal that CCK-8 and JMV-180 interact with different sites on the rat pancreatic acinar cell CCKA receptor
abstract: The ability of CCKA antagonists to inhibit full and partial CCK agonists of the rat pancreatic acinar cell CCKA receptor has been studied. When isolated rat pancreatic acini were superfused with CCK-8 (10 pM-1 nM) or CCK-4 (1 microM), an increase in [Ca2+]i signal was initiated. Concurrent superfusion of either L-364,718 (0.1 microM) or lorglumide (10 microM), chemically distinct, specific, potent antagonists of the CCKA receptor, resulted in a rapid inhibition of the [Ca2+]i signal initiated by all concentrations of CCK-8. In contrast, Ca2+ oscillations, initiated by JMV-180 (25 nM-1 microM), a partial agonist analogue of CCK-8, were essentially unaffected by concurrent superfusion of either L-364,718 or lorglumide. When JMV-179, an analogue of JMV-180 that exhibits characteristics of a pure antagonist, was superfused concurrently with either CCK-8 or JMV-180, Ca2+ oscillations were inhibited, even in the presence of 0.1 microM L-364,718. In a similar fashion, amylase secretion stimulated by CCK-8 was markedly attenuated by L-364,718, lorglumide, and JMV-179, whereas secretion stimulated by JMV-180 was only inhibited by JMV-179. A model is proposed to reconcile this data, based on the assumption that JMV-180 and CCK-8 interact with discrete sites on the CCKA receptor, which are differentially affected by the binding of antagonists. This model may also explain how a single receptor may transduce multiple signals in response to different agonists.

title: Electrophysiological properties and chemosensitivity of acutely isolated nodose ganglion neurons of the rabbit.
abstract: Neurons in the nodose ganglion were enzymatically dissociated from adult rabbits and electrophysiologically examined within 2 h. About 90% of the neurons survived the dissociation procedure. Their resting membrane potential (-59 +/- 0.6 mV), time constant (5.7 +/- 0.5 ms), input resistance (80 +/- 5.3 M omega), action potential properties (amplitude, 79 +/- 1.2 mV; overshoot 21 +/- 1.0 mV; duration 5.2 +/- 0.1 ms; threshold, -38 +/- 0.9 mV; and fast spike afterhyperpolarization, 10 +/- 0.5 mV, 64 +/- 3.8 ms) were not different from values obtained from nodose neurons in intact ganglion in vitro and in vivo. An unusually long duration spike afterhyperpolarization observed in about 35% of nodose neurons in intact ganglion was demonstrated in about 28% of the acutely isolated neurons. The percentage of isolated neurons responding to bath applied acetylcholine, bradykinin, serotonin and histamine was similar to those reported for neurons in the intact ganglia. We conclude that the electrophysiological and chemoreceptive properties of acutely isolated adult rabbit nodose neurons are similar to those observed for neurons in intact ganglia. Thus the availability of large numbers of acutely isolated adult nodose neurons should allow multidisciplinary studies of vagal afferent C-fiber neurons not readily obtainable in the intact ganglion.

title: Some membrane characteristics of single isolated nodose ganglion cells studied under current and voltage clamp conditions
abstract: The membrane characteristics of rabbit nodose ganglion neurons isolated by enzymatic digestion were investigated under current and voltage clamp conditions. The membrane of 5-10% of these cells appeared quite intact after enzymatic digestion and mechanical isolation, and this preparation can be successfully employed in biophysical studies. In fact, the membrane and action potential parameters of healthy cells were similar to those reported in whole ganglia or sliced preparations. Five distinct voltage-dependent membrane currents were recorded: fast inward, slow inward and slow outward currents elicited by depolarizing pulses; hyperpolarizing pulses elicited two currents, one associated with rectification and another occurring on the return of a hyperpolarizing pulse.

title: Cholecystokinin and neuropeptide Y receptors on single rabbit vagal afferent ganglion neurons: site of prejunctional modulation of visceral sensory neurons
abstract: A [125I]cholecystokinin (CCK) analog and [125I]peptide YY (PYY) were used to localize and characterize CCK and neuropeptide Y (NPY) receptor binding sites in the rabbit vagal afferent (nodose) ganglion. High concentrations of CCK and NPY binding sites were observed in 10.6% and 9.2% of the nodose ganglion neurons, respectively. Pharmacological experiments using CCK or NPY analogs suggest that both subtypes of CCK (CCK-A and CCK-B) and NPY (Y1 and Y2) receptor binding sites are expressed by discrete populations of neurons in the nodose ganglion. These results suggest sites at which CCK or NPY, released in either the nucleus of the solitary tract or a peripheral tissue, may modulate the release of neurotransmitters from a select population of visceral primary afferent neurons. Possible functions mediated by these receptors include modulation of satiety, opiate analgesia, and the development of morphine tolerance.

title: Characterization of cholecystokininA and cholecystokininB receptors expressed by vagal afferent neurons
abstract: The cholecystokinin receptors expressed by vagal afferent neurons mediate the effect of cholecystokinin in inhibiting food intake and gastric emptying. We have determined the relative abundance of cholecystokininA, gastrin-cholecystokininB and gastrin-cholecystokininC receptor populations in the rat vagus by autoradiography using [125I]Bolton Hunter-cholecystokinin-8, [125I]Bolton Hunter-heptadecapeptide gastrin and [125I]Leu(15)2-17Glycine-extended heptadecapeptide gastrin, together with the selective antagonists devazepide and L-740093. The results indicate approximately three-fold higher abundance of cholecystokininA compared with gastrin-cholecystokininB receptors, and no significant representation of gastrin-cholecystokininC receptors. Topical capsaicin applied to the vagal nerve trunk abolished the accumulation of sites binding both [125I]Bolton Hunter-labelled cholecystokinin-8 and heptadecapeptide gastrin indicating that both cholecystokininA and gastrin-cholecystokininB receptor populations were present on afferent fibres. The molecular identity of the receptors expressed by rat and human nodose ganglia was examined using the reverse transcription polymerase chain reaction. Products of the predicted size for the cholecystokininA and gastrin-cholecystokininB receptors were identified. The human and rat cholecystokininA receptor products were cloned and the sequences were found to be 99% homologous to those published for receptors expressed by rat pancreas and human gall bladder. We conclude that cholecystokininA and gastrin-colecystokininB receptors are synthesized by nodose ganglion cells, and that the receptor proteins are transported to the periphery along afferent fibres. While there is a clear role for vagal cholecystokininA receptors, the function of vagal afferent gastrin-cholecystokininB receptors remains to be determined.

title: Electrophysiological and autoradiographical evidence for cholecystokinin A receptors on rat isolated nodose ganglia.
abstract: The sulphated octapeptide, cholecystokinin (CCK-8S), is believed to be a neurotransmitter of vagal sensory neurones, and here the presence of functional receptors for CCK-8S in the rat vagus nerve has been investigated by electrophysiological and autoradiographic techniques. CCK-8S caused concentration-dependent depolarizations when superfused over the rat isolated nodose ganglion at 37 degrees C as measured by a silicone grease gap technique. Concentration-response curves to CCK-8S were shifted to the right by low concentrations of the CCKA receptor antagonist, Devazepide, but not by the CCKB receptor antagonist, L-365,260, data which indicate that receptors were of the CCKA subtype. Consistent with this notion, the CCKB agonist, unsulphated CCK-8, was without effect until high concentrations (> 1 microM) were used. A synthetic analogue of CCK-8S, D-Tyr25(Nle28,31)-CCK 25-33S, which has been reported to be more stable and peptidase-resistant than CCK-8S, was equipotent with CCK-8S in depolarizing the nodose ganglion. When D-Tyr25(Nle28,31)-CCK 25-33S was labelled with 125I, it bound to tissue sections of nodose ganglion. By light microscopic autoradiography, silver grains were found to be highly localized over cell bodies of vagal sensory neurones. An excess of CCK-8S inhibited binding as did Devazepide, but not L-365,260, confirming that binding sites were CCKA subtype receptors. These results indicate the existence of functional CCKA receptors in the nodose ganglion and strengthen the case for the involvement of vagal sensory neurones in gastric emptying and satiety.

title: Ca2+ oscillations in pancreatic acinar cells: spatiotemporal relationships and functional implications.
abstract: The pancreatic acinar cells are of particular interest for the study of cytosolic Ca2+ signals, since they are morphologically polarized and generate agonist-specific Ca2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca2+ signals and the Ca(2+)-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimic these responses. The shortlasting local Ca2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

title: Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells.
abstract: Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.

title: Effects of CCK-8 on the cytoplasmic free calcium concentration in isolated rat islet cells.
abstract: The C-terminal octapeptide of cholecystokinin (CCK-8) is known to stimulate insulin secretion. We examined its effects on the cytoplasmic free calcium concentration ([Ca2+]IC) in isolated rat pancreatic islet cells. At 8.3 mM glucose and 1.28 mM Ca2+, CCK-8 (100 nM) rapidly increased [Ca2+]IC to a short-lived peak, whereafter the [Ca2+]IC, within 1.5 minutes, fell to values below baseline. CCK-8 also rapidly increased the [Ca2+]IC at 3.3 mM glucose and in a calcium deficient medium. However, either at low glucose or in the absence of extracellular Ca2+, the post-peak [Ca2+]IC did not fall below baseline levels. The CCKA receptor antagonist, L-364,718 (20 nM), inhibited the effects of CCK-8 on [Ca2+]IC. The results suggest that CCK-8 in islet cells liberates calcium from intracellular stores by activating CCKA receptors.

title: Linkage of [Ca2+]i in single isolated D cells to somatostatin secretion induced by cholecystokinin.
abstract: The Ca2+/inositol phospholipid signaling cascade has been implicated in the mechanism by which cholecystokinin (CCK) stimulates gastric somatostatin release, but a direct linkage between intracellular events in gastric D cells and somatostatin secretion has not been established. To address this problem we developed a method for correlating somatostatin release with the measurement of intracellular Ca2+ concentration ([Ca2+]i) in isolated D cells.  at a peptide concentration of 2 x 10(-8) M. The CCK-mediated increase in [Ca2+]i was biphasic, with a rapid, initial transient elevation followed by a sustained plateau. The rise in [Ca2+]i was accompanied by a concomitant increase in release of somatostatin-like immunoreactivity (SLI). Removal of extracellular Ca2+ had no effect on the initial transient elevation in [Ca2+]i induced by CCK but abolished both the sustained plateau in [Ca2+]i and the release of SLI. The selective CCK antagonist L-364, 718 (10(-7) M) inhibited the effects of CCK on both [Ca2+]i and SLI release. The nonspecific Ca2+ channel blocker NiCl2 (10(-3) M) and the L-type Ca2+ channel blocker nifedipine inhibited the sustained rise in [Ca2+]i and the release of SLI but left the initial transient increase in [Ca2+]i unaltered. These results indicate that CCK-stimulated release of SLI from D cells in the gastric fundus is linked to influx of extracellular Ca2+ via L-type Ca2+ channels.

title: Cholecystokinin increases intracellular Ca2+ concentration in the Human JURKAT T Lymphocyte cell line.
abstract: We have recently demonstrated the presence of specific high-affinity cholecystokinin binding sites of the central type on the Human JURKAT T Lymphocyte Cell line. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]i) in response to CCK (cholecystokinin) peptides were measured in the Human JURKAT T Lymphocyte Cell line by fura-2 fluorometry. CCK-8 (the C-terminal octapeptide of CCK), the potent CCK analog Boc-[Nle28,31]CCK-7, stimulated ([Ca2+]i) mobilization in a dose-dependent manner in cells preloaded with fura-2 AM with an EC50 of 2.4 +/- 1 nM and 8 +/- 2 nM, respectively. The selective CCKB receptor agonists, namely Boc-Trp-Nle-Asp-Phe-NH2 and the cyclic analog JMV320,  see text], were also potent in stimulating mobilization of [Ca2+]i with an EC50 of 32 +/- 10 nM and 25 +/- 10 nM, respectively. Compound JMV180, Boc-Tyr(SO3H)-Nle-Trp-Nle-Asp-2-phenylethyl ester, did not stimulate [Ca2+]i but inhibited the mobilization of [Ca2+]i elicited by 10 nM CCK-8 in a dose-dependent manner with an IC50 of 10 +/- 2 nM. The selective non-peptide CCKB receptor antagonist L-365,260 was more potent than the selective CCKA receptor antagonist MK-329 in inhibiting the [Ca2+]i mobilization elicited by 10 nM CCK-8 with IC50 values of 20 +/- 8 nM and 400 +/- 100 nM, respectively. These data indicated that CCK-8 and potent CCK analogs induced [Ca2+]i mobilization in the Human JURKAT T cell line through the CCKB/gastrin receptor type.

title: Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.
abstract: A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988)  254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400

title: The cholecystokinin-induced increase in intracellular calcium in AR42J cells is mediated by CCKB receptors linked to internal calcium stores
abstract: Changes in intracellular levels of free [Ca2+]i were monitored in cell suspensions and either single cells or cell clusters of the rat pancreatic tumour cell line AR42J grown on cover slips. Increases in free [Ca2+]i were seen when the bathing medium contained cholecystokinin octapeptide sulphated (CCK) or CCKB receptor agonists. Responses to CCK agonists were repeatable and reversed on washout. The responses to cholecystokinin and pentagastrin could be blocked by selective CCKB receptor antagonists but not a CCKA receptor antagonist. Depleting internal Ca2+ stores with thapsigargin blocked the response to pentagastrin suggesting that the response was mediated by Ca2+ release from internal stores. The rapid run down of the pentagastrin response in the absence of extracellular Ca2+ shows that replenishment of internal stores by extracellular Ca2+ is important in maintaining the CCK response.

title: Effects of cholecystokinin on cytosolic calcium in pancreatic duct segments and ductal cells.
abstract: Although the gastrointestinal peptide cholecystokinin (CCK) has been shown to increase bicarbonate and water secretion and potentiate the effects of secretin on pancreatic ducts, CCK receptors have not been identified on pancreatic ductal epithelium. The effects of CCK octapeptide (CCK-8) on cytosolic calcium were evaluated on microscopically dissected rat pancreatic duct segments and single rat duct cells from the established ARIP cell line. By use of fluorescence microscopy in fura-2-loaded duct segments or single cells, intracellular calcium concentration was measured in response to CCK-8 (4 x 10(-12)-4 x 10(-8) M). CCK-8 significantly increased cytosolic calcium up to 50-fold over baseline. The greatest increase occurred with the highest concentration of CCK-8 (4 x 10(-8) M). Oscillations were observed in experiments performed in buffer containing 0.68 mM physiological calcium. In another series of experiments performed in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to deplete extracellular calcium, CCK-8 treatment still resulted in significant increases in cytosolic calcium; however, oscillations were abolished. Since cytosolic calcium increased in the absence of extracellular calcium, the initial calcium rise most likely was from cytosolic stores. Our findings of CCK-8-stimulated increases in cytosolic calcium in microdissected pancreatic ducts suggest the presence of CCK receptors, a characteristic that was not lost in cultured pancreatic ductal cells. In addition, in ARIP cells, the CCK-8-induced increase in cytosolic calcium was abolished by pretreatment with the selective CCK-B receptor antagonist L-365,260 but not by the CCK-A receptor antagonist L-364,718.

title: Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells.
abstract: The ability of cholecystokinin (CCK) to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. CCK-8 elevated the cytosolic Ca2+ levels in cell line NCI-H345 in a dose dependent manner. Nanomolar concentration of CCK-8 elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent CCK agonists such as gastrin-1 and nonsulfated CCK-8 but not inactive compounds such as CCK-27-32-NH2 elevated cytosolic Ca2+ levels. These data suggest that CCK receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.

title: Cholecystokinin increases intracellular Ca2+ concentration in cultured striatal neurons
abstract: Although it has been established that pancreatic cholecystokinin (CCK) receptors are coupled to phosphatidylinositol turnover, the events which follow activation of CCK receptors in the central nervous system have not received much attention. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]1) in response to CCK peptides were measured in single cultured rat striatal neuron by fura-2 fluorometry. CCK peptides dose-dependently increased [Ca2+]i in a monophasic manner. The order of the potencies of CCK peptides to increase [Ca2+]i was as follows: caerulein greater than CCK-8 greater than desulfated CCK-8 greater than CCK-4. The effect of caerulein was completely blocked in a Ca2(+)-depleted medium. In addition, omega-conotoxin GVIA completely inhibited the effect of caerulein, while neither nifedipine nor verapamil affected it. Our results indicate that CCK receptors couple to N-type voltage-sensitive Ca2+ channels in cultured rats striatal neurons.

title: Expression and regulation of cholecystokinin and cholecystokinin receptors in rat nodose and dorsal root ganglia
abstract: Cholecystokinin (CCK) is an important satiety factor, acting via the vagus nerve to influence central feeding centers. CCK binding sites have been demonstrated in the vagal sensory nodose ganglion and within the nerve proper. Using in situ hybridization, expression of the CCK(A) and (B) receptors (Rs), as well as of CCK itself, was studied in the normal nodose ganglion (NG), and after vagotomy, starvation and high-fat diet. CCK(A)-R mRNA expression in dorsal root ganglia (DRGs) was also explored. In the NG, 33% of the neuron profiles (NPs) contained CCK(A)-R mRNA and in 9% we observed CCK(B)-R mRNA. CCK mRNA was not found in normal NGs. Peripheral vagotomy decreased the number of CCK(A)-R mRNA-expressing NPs, dramatically increased the number of CCK(B)-R mRNA, and induced CCK mRNA and preproCCK-like immunoreactivity in nodose NPs. No significant differences in the number of NPs labelled for either mRNA species were detected following 48 h food deprivation or in rats fed a high-fat content diet. In DRGs, 10% of the NPs expressed CCK(A)-R mRNA, a number that was not affected by either axotomy or inflammation. This cell population was distinct from neurons expressing calcitonin gene-related peptide mRNA. These results demonstrate that the CCK(A)-R is expressed by both viscero- and somatosensory primary sensory neurons, supporting a role for this receptor as a mediator both of CCK-induced satiety and in sensory processing at the spinal level. The stimulation of CCK and CCK(B)-R gene expression following vagotomy suggests a possible involvement in the response to injury for these molecules.

title: Characterization of type A and type B CCK receptor binding sites in rat vagus nerve
abstract: We employed quantitative receptor autoradiography to analyze pharmacological properties of 125I-Bolton Hunter cholecystokinin (CCK-8)-labeled binding sites in sections of rat cervical vagus nerve that had been ligated 24 h prior to extraction. Binding densities were detected in segments of nerve proximal and distal to the ligature. Analysis was confined to proximal segments. Saturation and competitive binding studies were carried out using sulphated CCK-8 and two selective CCK receptor antagonists: MK-329, to define type-A (CCKA) binding sites; and, L-365,260, to define type-B (CCKB) binding sites. Sulphated CCK-8 was the most potent inhibitor of vagal 125I-CCK binding (IC50 = 2 nM). Nonlinear curve fitting analysis of the CCK binding data favored the presence of a single class of vagal CCK receptors (KDi = 1 nM). However, both MK-329 (IC50 = 18 nM) and L-365,260 (IC50 = 45 nM) competed for vagal 125I-CCK binding indicating the presence of CCKA and CCKB binding sites. Co-analysis of the antagonist binding data suggested that CCKA and CCKB receptors were transported in equal concentrations within the vagus. MK-329 bound with high affinity to CCKA sites (Ki = 3 nM) and low-affinity to CCKB sites (Ki = 462 nM) while L-365,260 bound with high affinity to CCKB sites (Ki = 10 nM) and low-affinity to CCKA sites (Ki = 775 nM). These same ligands were used to characterize the specificity of 125I-CCK binding in the medial and lateral divisions of the nucleus of the solitary tract (NTS), two regions innervated by primary vagal afferents carrying CCK receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Vagal sensors in the rat duodenal mucosa: distribution and structure as revealed by in vivo DiI-tracing
abstract: Results from functional studies point to the importance of chemoreceptive endings in the duodenum innervated by vagal afferents in the regulation of gastrointestinal functions such as gastric emptying and acid secretion, as well as in the process of satiation. In order to visualize the vagal sensory innervation of this gut segment, vagal afferents were selectively labeled in vivo by injecting the lipophilic carbocyanine dye DiI into either the left or the right nodose ganglion of young adult rats. Thick cryostat sections or whole-mounted peels of muscularis externa or submucosa of formalinfixed tissue were analyzed with conventional and/or confocal microscopy. In the mucosa, many DiI-labeled vagal afferent fibers were found with terminal arborizations mainly between the crypts and the villous lamina propria. In both areas, vagal terminal branches came in close contact with the basal lamina, but did not appear to penetrate it so as to make direct contact with epithelial cells. Labeled vagal afferent fibers in the villous and cryptic lamina propria were found to be in intimate anatomical contact with fibrocyte-like cells that may belong to the class of interstitial cells of Cajal, and with small granular cells that might be granulocytes or histiocytes. Although our analysis was not quantitative, and considering that labeling was unilateral and not complete, it appears that the overall density of vagal afferent mucosal innervation was variable; many villi showed no evidence for innervation while other areas had quite dense networks of arborizing terminal fibers in several neighboring villi. Analysis of separate whole-mounted muscularis externa and submucosa peels revealed the presence of large bundles of labeled afferent fibers running within the myenteric plexus along the mesenteric attachment primarily in an aboral direction, with individual fibers turning towards the antimesenteric pole, and either penetrating into the submucosa or forming the characteristic intraganglionic laminar endings (IGLEs). Although the possibility of individual fibers issuing collaterals to myenteric IGLEs and at the same time to mucosal terminals was not demonstrated, it cannot be ruled out. These anatomical findings are discussed in the context of absorptive mechanisms for the different macronutrients and the implication of enteroendocrine cells such as CCK-containing cells that may function as intestinal “taste cells”.

title: Topographic inventories of vagal afferents in gastrointestinal muscle
abstract: To inventory and characterize the two types of vagal afferents (both putative mechanoreceptors) in the muscle of the gastrointestinal tract, the authors injected wheat germ agglutinin‐horseradish peroxidase into the nodose ganglia of rats that had received unilateral ventral rhizotomies to eliminate efferents. The gut, from the oral esophagus to the distal colon, was divided into wholemounts, processed with tetramethylbenzidine, and surveyed to establish normative topographic maps of afferents. Vagal intraganglionic laminar endings (IGLEs) were ubiquitous, with concentrations varying on a longitudinal gradient (higher rostrally). This overall gradient was punctuated by denser condensations of endings in the oral esophagus, gastric corpus, and distal ileum. In regional specializations, IGLEs were fused into conspicuous, dense networks in the laryngeal esophagus and the antrum. Intramuscular arrays (IMAs) had restricted distributions, including the walls of the stomach and the sphincters throughout the gut. In the forestomach, a singular concentration of orthogonally crossed IMAs was organized into a lattice or “fovea.” IMAs displayed variations in morphology, with one specialization consisting of short, terminal processes associated with sphincters and a more widespread form consisting of long, rectilinear processes in the forestomach, along the greater curvature, and in limited intestinal regions. On the basis of their topographic patterns and structural specializations, the two putative mechanoreceptors may have different functions: IGLEs appear situated to integrate intramural tension, and perhaps myenteric neuronal activity, into rhythmical, propagated motor programs, such as swallowing, peristalsis, and emptying. IMAs are distributed strategically and appear to satisfy structural requirements for stretch receptors tuned to tonic or more aperiodic events that may affect central nervous system processing as well as local gastrointestinal coordination.  2000.

title: Vagal afferents innervating the gastrointestinal tract and CCKA‐receptor immunoreactivity
abstract: A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA‐receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA‐receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C‐terminal (Ab‐1) nor an N‐terminal (Ab‐2) specific antibody produced any specific staining; in the myenteric plexus, non‐vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR‐immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR‐immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab‐1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR‐immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA‐receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals. Anat Rec 266:10–20, 2002.

title: Expression of cholecystokinin A receptors in neurons innervating the rat stomach and intestine.
abstract: Two distinct receptors, cholecystokinin (CCK)-A and CCK-B, mediate CCK effects in the digestive system. The aim of this study was to elucidate the cellular sites of expression of CCK-A receptor in the rat stomach and small intestine.



We developed and characterized an antibody to the N-terminal region (LDQPQPSKEWQSA) of rat CCK-A receptor and used it for localization studies with immunohistochemistry.



Specificity of the antiserum was demonstrated by (1) detection of a broad band at 85-95 kilodaltons in Western blots of membranes from CCK-A receptor CHO-transfected cells; (2) cell surface staining of CCK-A receptor-transfected cells, (3) translocation of CCK-A receptor immunostaining in CCK-A receptor-transfected cells after exposure to CCK; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with the peptide used for immunization. CCK-A receptor immunoreactivity was localized to myenteric neurons and to fibers in the muscle and mucosa. In the stomach, myenteric neurons and mucosal fibers were abundant. Many CCK-A receptor myenteric neurons contained the inhibitory transmitter vasoactive intestinal polypeptide, and some were immunoreactive for the excitatory transmitter substance P. Subdiaphragmatic vagotomy reduced the density of CCK-A receptor fibers in the gastric mucosa by approximately 50%, whereas celiac/superior mesenteric ganglionectomy had no detectable effect on fiber density.



CCK-A receptor is expressed in functionally distinct neurons of the gastrointestinal tract. CCK-A receptor may mediate reflexes stimulated by CCK through the release of other transmitters from neurons bearing the receptor.

title: The role of endogenous cholecystokinin in the sensory transduction of luminal nutrient signals in the rat jejunum
abstract: Some vagal afferent fibres are exquisitely sensitive to exogenous administration of cholecystokinin (CCK) but their sensitivity to endogenous CCK released by luminal stimuli has not been demonstrated directly, although implied from reflex and behavioural studies. We have therefore utilised electrophysiological techniques to record afferent discharge in mesenteric nerve bundles supplying the rat jejunum in response to luminal application of casein acid hydrolysate (CAH). CAH stimulated whole nerve afferent discharge in both in vivo and in vitro preparations (P < 0.01) while single unit analysis revealed that fibres sensitive to CAH also responded to exogenous CCK. The responses to both CCK and CAH were abolished by the CCKA antagonist devazepide. This study therefore supports the hypothesis that a functional relationship exists between CCK-containing enteroendocrine cells and the afferent fibres whose terminals lie within close proximity.