Capsaicin treatment destroys vagal afferent C fibers and markedly attenuates reduction of food intake and induction of hindbrain Fos expression by CCK. However, both anatomical and electrophysiological data indicate that some gastric vagal afferents are not destroyed by capsaicin. Because CCK enhances behavioral and electrophysiological responses to gastric distension in rats and people, we hypothesized that CCK might enhance the vagal afferent response to gastric distension via an action on capsaicin-insensitive vagal afferents. To test this hypothesis, we quantified expression of Fos-like immunoreactivity (Fos) in the dorsal vagal complex (DVC) of capsaicin-treated (Cap) and control rats (Veh), following gastric balloon distension alone and in combination with CCK injection. In Veh rats, intraperitoneal CCK significantly increased DVC Fos, especially in nucleus of the solitary tract (NTS), whereas in Cap rats, CCK did not significantly increase DVC Fos. In contrast to CCK, gastric distension did significantly increase Fos expression in the NTS of both Veh and Cap rats, although distension-induced Fos was attenuated in Cap rats. When CCK was administered during gastric distension, it significantly enhanced NTS Fos expression in response to distension in Cap rats. Furthermore, CCK's enhancement of distension-induced Fos in Cap rats was reversed by the selective CCK-A receptor antagonist lorglumide. We conclude that CCK directly activates capsaicin-sensitive C-type vagal afferents. However, in capsaicin-resistant A-type afferents, CCK's principal action may be facilitation of responses to gastric distension.

The pancreas and gastrointestinal tract (GIT) of adults and of an embryonic stage of 11 cm long (about half the length of newborn fish) of the spiny dogfish, Squalus acanthias, were investigated immunocytochemically for the occurrence of the gastro-entero-pancreatic (GEP) neurohormonal peptides. In the pancreas of adult forms 5 endocrine cell types were seen, namely insulin-, somatostatin-, glucagon-, pancreatic polypeptide (PP)- and gastric inhibitory peptide (GIP)-immunoreactive cells. These cell types form scattered islets and were seen sometimes to surround small ducts. GIP-immunoreactivity cells did not occur in glucagon-containing cells. In the mucosa of GIT of adults 18 endocrine cell types were observed, viz. insulin-, somatostatin-, glucagon-, glicentin, PP-, polypeptide YY (PYY)-, vasoactive intestinal polypeptide (VIP)-, GIP-, gastrin C-terminus, CCK-, neurotensin N-terminus-, bombesin/gastrin releasing peptide (GRP)-, substance P-, enkephalin-, alpha-endorphin, beta-endorphin-, serotonin- and calcitonin immunoreactive cells. These cells occurred mostly in the intestine. All these cell types were of the open type, except glucagon- and glicentin-immunoreactive cells in the stomach, which seemed to be of the closed type. In the muscle layers and the submucosa, VIP and substance P- immunoreactive nerves and neurons were observed. In the pancreas of the dogfish embryo only 3 endocrine cell types could be demonstrated, namely insulin-, somatostatin- and glucagon-immunoreactive cells. In the mucosa of the GIT of the embryos studied 12 endocrine cell types were detected, viz. insulin-, somatostatin-, glucagon-, PP-, PYY-, VIP, GIP, gastrin C-terminus-, CCK-, neurotensin N-terminus-, enkephalin- and serotonin immunoreactive cells. The number of these cells, except that of PYY-immunoreactive cells, was lower than that of adults and in some cases their distribution did not correspond with that of adults.

The hypothesis that endogenous cholecystokinin (CCK) released from the small intestine during feeding causes satiety was tested in rat pups, 9 to 12 days old. Intragastric administration of soybean trypsin inhibitor, a procedure that releases CCK from the small intestine, decreased the subsequent intake of a test meal. This effect was reversed by prior treatment with MK-329, a selective antagonist of CCK at alimentary-type CCK (CCK-A) receptors. Thus, endogenous, small intestinal CCK can cause satiety in the neonatal rat and this effect involves CCK-A receptors.

Using whole cell patch clamp in thin brain stem slices, we tested the effects of cholecystokinin (CCK) on identified gastric-projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Perfusion with the sulfated form of CCK octapeptide (CCKCCKCCKCCKCCKCCKCCK-A receptor antagonist lorglumide (0.3-3 microM) attenuated the CCKCCK-B antagonist triglumide did not attenuate the CCKCCKCCK-A receptors were localized on the membrane of 34, 65, and 60% of fundus-, corpus-, and antrum/pylorus-projecting DMV neurons, respectively. Our data indicate that CCK-A receptors are present on a subpopulation of gastric-projecting neurons and that their activation leads to excitation of the DMV membrane.

Human gallbladders with cholesterol stones (ChS) exhibit an impaired muscle contraction and relaxation and a lower CCK receptor-binding capacity compared with those with pigment stones (PS). This study was designed to determine whether there is an abnormal receptor-G protein coupling in human gallbladders with ChS using (35)S-labeled guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, (125)I-labeled CCK-8 autoradiography, immunoblotting, and G protein quantitation. CCK and vasoactive intestinal peptide caused significant increases in [(35)S]GTPgammaS binding to Galpha(i-3) and G(s)alpha, respectively. The binding was lower in ChS than in PS (P < 0.01). The reduced [(35)S]GTPgammaS binding in ChS was normalized after the muscles were treated with cholesterol-free liposomes (P < 0.01). Autoradiography and immunoblots showed a decreased optical density (OD) for CCK receptors, an even lower OD value for receptor-G protein coupling, and a higher OD for uncoupled receptors or Galpha(i-3) protein in ChS compared with PS (P < 0.001). G protein quantitation also showed that there were no significant differences in the Galpha(i-3) and G(s)alpha content in ChS and PS. We conclude that, in addition to an impaired CCK receptor-binding capacity, there is a defect in receptor-G protein coupling in muscle cells from gallbladder with ChS. These changes may be normalized after removal of excess cholesterol from the plasma membrane.

Appetite suppressants may be one strategy in the fight against obesity. This study evaluated whether Korean pine nut free fatty acids (FFA) and triglycerides (TG) work as an appetite suppressant. Korean pine nut FFA were evaluated in STC-1 cell culture for their ability to increase cholecystokinin (CCK-8) secretion vs. several other dietary fatty acids from Italian stone pine nut fatty acids, oleic acid, linoleic acid, alpha-linolenic acid, and capric acid used as a control. At 50 muM concentration, Korean pine nut FFA produced the greatest amount of CCK-8 release (493 pg/ml) relative to the other fatty acids and control (46 pg/ml). A randomized, placebo-controlled, double-blind cross-over trial including 18 overweight post-menopausal women was performed. Subjects received capsules with 3 g Korean pine (Pinus koraiensis) nut FFA, 3 g pine nut TG or 3 g placebo (olive oil) in combination with a light breakfast. At 0, 30, 60, 90, 120, 180 and 240 minutes the gut hormones cholecystokinin (CCK-8), glucagon like peptide-1 (GLP-1), peptide YY (PYY) and ghrelin, and appetite sensations were measured. A wash-out period of one week separated each intervention day.CCK-8 was higher 30 min after pine nut FFA and 60 min after pine nut TG when compared to placebo (p < 0.01). GLP-1 was higher 60 min after pine nut FFA compared to placebo (p < 0.01). Over a period of 4 hours the total amount of plasma CCK-8 was 60% higher after pine nut FFA and 22% higher after pine nut TG than after placebo (p < 0.01). For GLP-1 this difference was 25% after pine nut FFA (P < 0.05). Ghrelin and PYY levels were not different between groups. The appetite sensation "prospective food intake" was 36% lower after pine nut FFA relative to placebo (P < 0.05). This study suggests that Korean pine nut may work as an appetite suppressant through an increasing effect on satiety hormones and a reduced prospective food intake.

OBJECTIVE

Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis.

METHODS

Cultured cardiac fibroblasts (CFs) were activated by TGF-β1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson's trichrome staining.

RESULTS

miR-223 was increased in CFs compared to cardiomypcytes, and TGF-β1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-β1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3' untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis.

CONCLUSIONS

miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.

As in a previous study (Biochim, Biophys. Acta 1224 (1994) 127-138), we used quantitative immunoblot analysis and found that rat pancreatic acini possess four different isoforms of PKC-alpha, delta, epsilon and zeta. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) caused translocation of each isoform from the cytosol to the membrane fraction. CCK-8 increased diacylglycerol (DAG) and caused translocation of PKC-sigma and PKC-epsilon but not that of PKC-alpha or PKC-zeta. L-364,718, a CCK receptor antagonist, prevented as well as reversed the effects of CCK-8 on DAG and on translocation of PKC-sigma and PKC-epsilon. To explore the possibility that different isoforms of PKC might have different distributions in rat pancreas, we used immunocytochemistry to determine the cellular distribution of different isoforms of PKC in intact pancreas as well as pancreatic acini. In intact pancreas, PKC-alpha and PKC-sigma were detected in islet cells but not in duct or acinar cells. PKC-epsilon was detected in the apical region of acinar cells and PKC-zeta was detected over the luminal surfaces of acinar cells and the ductules that extend from the acinus. Neither PKC-epsilon nor PKC-zeta was detected in islets. In pancreatic acini PKC-alpha and PKC-sigma were detected in islets or fragments of islets that contaminated the preparation but were not detected in acinar cells. PKC-epsilon was detected in the apical region of acinar cells and adding 1 microM TPA or 1 microM CCK-8 accentuated the immunostaining but did not alter its cellular distribution. L-364,718 reversed the changes in immunostaining caused by CCK-8. PKC-zeta was detected over the luminal surface of the acinar cells. TPA, but not CCK-8 or CCK-8 followed by L-364,718, increased the number of acini that showed staining of the luminal surfaces of acinar cells. Thus, the present results demonstrate that different isoforms of PKC are distributed differently in rat pancreas and that the different patterns of distribution can explain, at least in part, the different responses to CCK-8.

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.

Cholecystokinin (CCK) receptor binding was measured in rodent and primate brain and spinal cord using 125I-Bolton Hunter CCK-8 (125I-BH-CCK) and the selective non-peptide CCK antagonists MK-329 and L-365,260. In homogenate binding studies, L-365,260 displayed nanomolar affinity for CCK-B receptors in the cerebral cortex of several species including man (pIC50 congruent to 8.2) but showed low affinity for CCK-A receptors in the rat pancreas (pIC50 congruent to 6.3). By contrast, the CCK-A antagonist MK-329 showed the reverse selectivity (cortex: pIC50 congruent to 6.9, pancreas: pIC50 = 9.6). In autoradiographs of rat and monkey brain. 125I-BH-CCK binding was localized regionally with high levels being detected in the cerebral cortex, basal ganglia and some mid- and hindbrain nuclei. Specific 125I-BH-CCK binding was also localized to the substantia gelatinosa of the rat, monkey and human spinal cord. L-365,260 inhibited binding to most areas of the brain, but in the rat medial nucleus tractus solitarii and the monkey nucleus tractus solitarii. dorsomedial nucleus and infundibular hypothalamic nuclei together with the dorsomedial aspects of the caudate nucleus, where CCK-A sites are present, L-365,260 failed to displace all 125I-BH-CCK binding. In the primate spinal cord, L-365,260 was a relatively weak inhibitor of 125I-BH-CCK binding (pIC50 congruent to 6.0) whereas MK-329 showed high affinity for the CCK-A sites present there (pIC50 congruent to 9.6).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that CCK participates in the control of meal size is compelling, but the avenues by which CCK may affect daily food intake and body weight regulation are still uncertain. Although participation of brain CCK in control of food intake is acknowledged, our focus here is on participation of peripheral CCK in the control of food intake. Therefore, in this article we (1) review evidence for CCK's participation in control of meal size, (2) document involvement of CCK-A receptors located on vagal sensory neurons in control of food intake by exogenous and endogenous CCK, (3) point out apparent discrepancies in the experimental record, which auger for non-endocrine sources of CCK and non-vagal sites of CCK action, and (4) summarize recent observations, suggesting mechanisms by which CCK could participate in the control of daily food intake and body weight regulation.

This electrophysiological study was performed to elucidate the interactions of serotonin (5-hydroxytryptamine, 5-HT) and cholecystokinin (CCK) on mesenteric afferents supplying the rat jejunum. 5-HT and CCK produced characteristic responses in multi-unit recordings of mesenteric afferents. Waveform analysis to extract single units from the whole nerve recording identified populations of single afferents that were sensitive to either 5-HT or CCK, but not both. Furthermore, devazepide (0.5 mg/kg) completely abolished the response to CCK without altering the response to 5-HT while granisetron (0.5 mg/kg) abolished the response to 5-HT with no effect on the response to CCK. These results suggest that there are discrete, noninteractive populations of jejunal afferents that possess either 5-HT3 or CCK-A receptors but not both.

The paradigm for the control of feeding behavior has changed significantly. Research has shown that leptin, in the presence of CCK, may mediate the control of short-term food intake. This interaction between CCK and leptin occurs at the vagus nerve. In the present study, we aimed to characterize the interaction between CCK and leptin in the vagal primary afferent neurons. Single neuronal discharges of vagal primary afferent neurons innervating the gastrointestinal tract were recorded from rat nodose ganglia. Three groups of nodose ganglia neurons were identified: group 1 responded to CCK-8 but not leptin; group 2 responded to leptin but not CCK-8; group 3 responded to high-dose CCK-8 and leptin. In fact, the neurons in group 3 showed CCK-8 and leptin potentiation, and they responded to gastric distention. To identify the CCK-A receptor (CCKAR) affinity states that colocalize with the leptin receptor OB-Rb, we used CCK-JMV-180, a high-affinity CCKAR agonist and low-affinity CCKAR antagonist. As expected, immunohistochemical studies showed that CCK-8 administration significantly potentiated the increase in the number of c-Fos-positive neurons stimulated by leptin in vagal nodose ganglia. Administration of CCK-JMV-180 eliminated the synergistic interaction between CCK-8 and leptin. We conclude that both low- and high-affinity CCKAR are expressed in nodose ganglia. Many nodose neurons bearing low-affinity CCKAR express OB-Rb. These neurons also respond to mechanical distention. An interaction between CCKAR and OB-Rb in these neurons likely facilitates leptin mediation of short-term satiety.

1. Coexisting with oxytocin or vasopressin in the cell bodies and nerve terminals of the hypothalamic-neurohypophysial system are smaller amounts of other peptides. For a number of these "copeptides" there is strong evidence of corelease with the major magnocellular hormones. Guided by the location of their specific receptors we have studied the effects of three copeptides, dynorphin, cholecystokinin (CCK), and corticotropin releasing hormone (CRH), on the secretion of oxytocin and vasopressin from isolated rat neural lobe or neurointermediate lobe preparations in vitro. 2. Dynorphin is coreleased with vasopressin from neural lobe nerve terminals and acts on neural lobe kappa-opiate receptors to inhibit the electrically stimulated secretion of oxytocin. Naloxone augments oxytocin release from the neural lobe in a manner directly proportional to the amount of vasopressin (and presumably dynorphin) released. 3. Cholecystokinin, coreleased with oxytocin by neural lobe terminals, has been shown to have high-affinity receptors located in the NL and to stimulate secretion of both oxytocin and vasopressin. CCK's secretagogue effect was independent of electrical stimulation and extracellular Ca2+ and was blocked by an inhibitor of protein kinase C. 4. CRH, coreleased with OT from the neural lobe, has receptors in the intermediate lobe of the pituitary, but not in the neural lobe itself. CRH stimulates the secretion of oxytocin and vasopressin from combined neurointermediate lobes but not from isolated neural lobes. Intermediate lobe peptides, alpha and gamma melanocyte stimulating hormone, induced secretion of oxytocin and vasopressin from isolated neural lobes. Their effect was, like that of CCK, independent of electrical stimulation and extracellular Ca2+ and blocked by an inhibitor of protein kinase C. 5. Among the CRH-producing parvocellular neurons of the paraventricular nucleus, in the normal rat, approximately half also produce and store vasopressin. After removal of glucocorticoid influence by adrenalectomy, virtually all of the CRH neurons contain vasopressin. 6. The two subtypes of CRH neurosecretory cells found in the normal rat possess different topographical distributions in the paraventricular nucleus, suggesting the possibility of differential innervation. Stress selectively activates the vasopressin containing subpopulation of CRH neurons, indicating that there are separate channels of regulatory input controlling the two components of the parvocellular CRH neurosecretory system.

Exocrine pancreatic cancer is significantly more common in younger men than in younger women. The male-to-female sex ratio is, in most countries, between 1.25 and 1.75 to 1, but decreases with increasing age. Moreover, prior oophorectomy appeared in one study to be significantly more common in women with pancreatic cancer than in controls. This has raised interest in sex hormones in the development in pancreatic cancer. It has been questioned if there are estrogen receptors in ductal pancreatic cancer, but there are no doubt estrogen receptors and estrogen-binding protein in human healthy pancreas. It is also well proven that it is possible to influence experimental pancreatic cancer with estrogens. However, in clinical studies tamoxifen has repeatedly been shown to be without significant effects. On the other hand, there are also androgen receptors in pancreatic cancer and testosterone has been shown to strongly promote growth in experimental pancreatic cancers. It is therefore of considerable interest that an antiandrogen recently was shown to significantly prolong life in patients with unresectable pancreatic carcinoma. However, in patients with advanced pancreatic carcinoma the S-testosterone is low, far lower than what could be expected due to weight-loss and malnourishment alone. Pancreatic cancer has etiologically been connected to diet, for example the intake of fat. Cholecystokinin receptors have been found on human pancreatic cancer, possible to stimulate in vitro by cholecystokinin (CCK). Studies with CCK-receptor binding, hybridization with radiolabeled complementary DNA (cDNA) probes, or reverse-transcription polymerase chain reaction have shown that CCK-A receptors also are present in rat pancreatic putative preneoplastic lesions and cancer tissue, rat pancreatic-cancer cell lines, pancreatic carcinomas in transgenic mice, hamster pancreatic cancer, and human pancreatic cancer cell lines and tumors. Also, CCK-B receptors have been found in some human pancreatic cancers. There are a vast number of experiments done on CCK-stimulation of pancreatic cancer. They indicate that CCK may have a promotional effect on exocrine pancreatic cancer, but it is not probable that hyperstimulation with CCK alone induce pancreatic cancer. At present, however, despite a lot of evidence for a hormone-dependence of pancreatic cancer there are no data confirming a role for estrogens, androgens, CCK or their antagonists in clinical treatment of exocrine pancreatic cancer.

Opioid effectiveness can be improved by individualizing dosing, route of administration and the drug. Particularly in the treatment of chronic non-cancer pain, careful patient selection is essential. The current review concentrates on new ideas about improving opioid effectiveness by increasing efficacy or reducing adverse effects by combining other drugs that modulate opioid receptor mediated effects. These pharmacological "oipioid adjuvants" include e.g. alpha(2)-adrenergic agonists, non-steroidal anti-flammatory analgesics, NMDA-receptor antagonists, CCK-antagonists, gabapentinoids and NK-1 receptor antagonists. The theoretical background and the clinical evidence of these combinations will be discussed.

BACKGROUND

Gastrin and cholecystokinin (CCK) are thought to exert trophic effects on the gastrointestinal tract and pancreas. Two types of receptors have been cloned, CCK-A and CCK-B/ gastrin. We have examined the occurrence of CCK-A and CCK-B receptor mRNA in the brain, digestive tract, pancreas, and kidney of the rat and man by Northern blot and reverse transcribed polymerase chain reaction (RT-PCR).

METHODS

Total RNA was isolated from rat tissues and reverse transcribed into cDNA. cDNA from brain, kidney, and pancreas of the rat and man and from human whole stomach were commercially available. Northern blot and a PCR technique based on Taq polymerase-antibody interaction and using CCK-A and CCK-B receptor-specific primers, followed by Southern blot analysis, were the methods used.

RESULTS

By means of Northern blots, CCK-A receptor mRNA was detected in rat fundus mucosa and pancreas but not in the remaining GI tract or brain. By means of RT-PCR, CCK-A receptor mRNA was demonstrated in the brain and the mucosa of the fundus, antrum, duodenum, and colon, kidney, pancreas and pancreatic islets. CCK-B receptor mRNA was detected by Northern blot analysis in the brain and the fundus mucosa but not in the rest of the digestive tract and not in the pancreas, pancreatic islets, or kidney. By RT-PCR, expression of CCK-B receptor mRNA could also be detected in antrum mucosa. In man, CCK-A receptor mRNA was detected in the brain, stomach, pancreas, and kidney, whereas CCK-B receptor mRNA was found in the brain, stomach, and pancreas but not in the kidney. Cloning and DNA-sequence analysis of the PCR-amplified rat and human CCK-A and CCK-B receptor DNA fragments, which cover the protein-encoding regions of the intracellular loop C3, showed complete sequence homology as compared with published rat and human sequences.

CONCLUSIONS

It appears unlikely that CCK will have effects in the ileum, at least not effects mediated by CCK-A receptors. It also appears unlikely that physiologic concentrations of gastrin in the circulation will promote growth (or exert other effects) in the pancreas, duodenum, ileum, and colon, since CCK-B receptor mRNA is not expressed or is poorly expressed in these tissues.

Gastrin and cholecystokinin (CCK) act as growth factors for the gastric mucosa and the pancreas, respectively. CCK is also responsible, via the CCK-A receptor, for the pancreatic hyperplasia observed following the feeding of protease inhibitors or pancreaticobiliary diversion. Hypergastrinaemia does not increase the incidence of spontaneous gastrointestinal carcinoma, but does stimulate the proliferation of gastric enterochromaffin-like cells via the gastrin/CCK-B receptor, with a consequent increase in the incidence of gastric carcinoids. Whether gastrin influences mutagen-induced gastrointestinal carcinogenesis is still controversial, but CCK clearly enhances the induction by carcinogens of acinar tumours in the pancreas. While gastrin increases xenograft growth of 50% of gastrointestinal tumours tested, effects on the proliferation of gastrointestinal tumour cell lines in vitro have been more difficult to demonstrate, perhaps because many cell lines are already maximally stimulated by autocrine gastrin. Gastrin mRNA and progastrin, but not mature amidated gastrin, have been detected in all gastrointestinal cell lines tested. Although cell proliferation is inhibited by gastrin/CCK receptor antagonists, the spectrum of antagonist affinities is not consistent with binding to either CCK-A or gastrin/CCK-B receptors. Definition of the molecular structure of the receptor involved in the autocrine loop may lead to novel therapies for gastrointestinal cancer.

The actions of cholecystokinin (CCK) on gastrointestinal functions occur mainly via paracrine effects on peripheral sensory vagal fibers, which engage vago-vagal brain stem circuits to convey effector responses back to the gastrointestinal tract. Recent evidence suggests, however, that CCK also affects brain stem structures directly. Many electrophysiological studies, including our own, have shown that brain stem vagal circuits are excited by sulfated CCK (CCK-8s) directly, and we have further demonstrated that CCK-8s induces a remarkable degree of plasticity in GABAergic brain stem synapses. In the present study, we used fasted, anesthetized Sprague-Dawley rats to investigate the effects of brain stem administration of CCK-8s on gastric tone before and after activation of the esophageal-gastric reflex. CCK-8s microinjected in the dorsal vagal complex (DVC) or applied on the floor of the fourth ventricle induced an immediate and transient decrease in gastric tone. Upon recovery of gastric tone to baseline values, the gastric relaxation induced by esophageal distension was attenuated or even reversed. The effects of CCK-8s were antagonized by vagotomy or fourth ventricular, but not intravenous, administration of the CCK-A antagonist lorglumide, suggesting a central, not peripheral, site of action. The gastric relaxation induced by DVC microinjection of CCK-8s was unaffected by pretreatment with systemic bethanecol but was completely blocked by NG-nitro-L-arginine methyl ester, suggesting a nitrergic mechanism of action. These data suggest that 1) brain stem application of CCK-8s induces a vagally mediated gastric relaxation; 2) the CCK-8s-induced gastric relaxation is mediated via activation of nonadrenergic, noncholinergic pathways; and 3) CCK-8s reverses the esophageal-gastric reflex transiently.

Receptors for cholecystokinin (CCK), gastrin, neurotensin, somatostatin and vasoactive intestinal peptide (VIP) are over-expressed in several human tumors, where they have diagnostic and therapeutic implications. Since reports on the expression of these peptide receptors in primary gastric and colonic adenocarcinomas are either non-existent or conflicting, a detailed evaluation with particular emphasis on the tissue localization was undertaken. CCK-A, CCK-B, neurotensin, somatostatin and VIP receptors were localized by in vitro receptor autoradiography with iodinated radioligands on histological sections of surgical samples of 27 gastric and 25 colonic adenocarcinomas. CCK-A, CCK-B and neurotensin-1 receptors were found in a minority of both tumor types. Somatostatin receptors were found in 18/27 gastric and 2/25 colonic cancers. VIP receptors were found in 14/26 gastric and 23/25 colonic cancers; subtype characterization suggests VIP1 receptors. In addition, resected tumor samples contained non-malignant tissues (mucosa, smooth muscle, nerves or vessels) with high amounts of the various peptide receptors. Therefore, regulatory peptide receptors are expressed differentially in gastric and colonic cancers but also very frequently in "contaminating" non-malignant tissues. Since results using morphological techniques are superior to those using homogenates, we recommend that localization of these receptors to the tissues should always be attempted, to minimize receptor over-estimation in tumors and to prevent spurious results.

OBJECTIVE

Gastrin (G-17) stimulates the growth of certain gastric and colon cancers mostly through gastrin/cholecystokinin (CCK)-B receptors. Glycine-extended gastrin (Gly-G) stimulates growth of a rat pancreatic acinar cell line; however, the effect of Gly-G on human gastric cancers is not known. The purpose of this study was to characterize the trophic effect of G-17 and Gly-G on two human gastric cancer cell lines, AGS and SIIA.

METHODS

Binding analyses were performed, and cell growth was assessed by counting cells over a time course.

RESULTS

G-17 stimulated growth of both AGS and SIIA cells. In AGS cells, gastrin/CCK-B receptor antagonists inhibited the effect of G-17 and competitively antagonized 125I-G-17 binding, whereas the CCK-preferring (CCK-A) receptor antagonists had no effect. In contrast, CCK-A receptor antagonists inhibited the stimulatory effect of G-17 in SIIA cells, whereas CCK-B receptor antagonists had no effect. Gly-G stimulated the growth of AGS and SIIA cells; neither the CCK-B nor the CCK-A receptor antagonists blocked this effect.

CONCLUSIONS

G-17 stimulates proliferation of AGS cells through the CCK-B receptor; however, G-17-mediated growth of SIIA acts through a CCK-A-like receptor. Furthermore, Gly-G stimulates growth of human gastric cancer cell lines, possibly through a receptor other than the CCK-B or CCK-A receptor.

The influence of ovarian cycling and of exogenous estradiol on the cholecystokinin (CCK) satiety-signalling system was investigated in intact and ovariectomized Long-Evans rats, respectively. Intraperitoneal injection of 1 mg/kg devazepide, the most potent and selective CCK(A) receptor antagonist, increased test meal size during estrus, but not during diestrus, confirming the influence of hypothalamic-pituitary-gonadal function on CCK satiety in intact rats. Devazepide was then tested in ovariectomized rats that received chronic cyclic estradiol (2 microg estradiol benzoate on Tuesday and Wednesday each week) or oil treatment. Devazepide did not increase meal size in estradiol-treated rats on Tuesday, prior to estradiol treatment, compared to oil-treated rats, but did selectively increase meal size on Friday, late in the estradiol replacement cycle, compared to Tuesday, early in the cycle. These results suggest that a phasic potentiation of the endogenous CCK satiety-signalling system is part of the mechanism for the decrease in meal size in female rats during estrus.

The role of the vagal and spinal afferent innervation in the inhibition of gastric emptying induced by duodenal perfusion with a disaccharide (maltose 300 mM) was investigated in awake rats fitted with gastric and duodenal cannulas. Perfusion of the duodenum with maltose inhibited gastric emptying by 44%. Maltose-induced inhibition of gastric emptying was reversed by 46% and 100% by functional ablation of the vagal or spinal capsaicin-sensitive afferent innervation, respectively. Pretreatment with the specific CCK 'A' receptor antagonist, MK329, completely abolished the effects of maltose on gastric emptying. These results suggest that disaccharides inhibit gastric emptying via activation of capsaicin-sensitive afferents in the duodenal mucosa via a mechanism involving an action of CCK at type 'A' receptors, possibly located on afferent fibers.

PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.