Ultrastructural localization of cholecystokinin in endocrine cells of the dog duodenum by the immunogold technique

Cholecystokinin (CCK) has been localized by the immunogold technique in a type of endocrine cell of the dog duodenum characterized by small (166 +/- 38 nm) secretory granules with fairly dense, homogeneous core separated from its enveloping membrane by a thin clear space. The CCK cell is immunocytochemically distinct and cytologically different from other types of endocrine cells, as the secretin, GIP and motilin cells, already identified in the dog duodenum.     

The effect of total parenteral nutrition in the rat on a sub-group of enteroendocrine cells

The effect of total parenteral nutrition (TPN) in rats on a number of enteroendocrine cells was investigated. The rats were given a continuous intravenous infusion of basal TPN solution for 7 days. Samples from duodenum, jejunum and ileum were collected, immunostained and the immunoreactive cells quantified using a computerised morphometrics system. The endocrine cells containing somatostatin, cholecystokinin (CCK), gastric inhibitory polypeptide (GIP), neurotensin and enteroglucagon were investigated. The results demonstrated a significant reduction in the number of CCK cells in the duodenum and jejunum. In the ileum the neurotensin-immunoreactive cells were significantly increased in number (P less than 0.02). No change was seen in the number of cells immunostained for somatostatin, GIP or enteroglucagon. These data indicate that short term TPN has a definite effect on the enteroendocrine cell population which may be linked to the side effects of TPN seen in man.     

Distribution of gastrin and CCK cells in the rat gastrointestinal tract

Summary Gastrin and cholecystokinin (CCK) cells of the rat gastrointestinal tract have been studied by immunocytochemistry and radioimmunoanalysis. With antisera directed against the COOH-terminal tetrapeptide sequence, which is common to gastrin and CCK, three distinct endocrine cell types are detected. One of the cell types predominates in the antrum, is scarce in the rest of the gut and corresponds to the gastrin cell. The second cell type is virtually confined to the duodenum and jejunum and corresponds to the CCK cell. The third cell type occurs disseminated in the small intestines, predominates in the ileum, and reacts with COOH-terminus-specific antisera only following diethylpyrocarbonate and not following formaldehyde fixation. It is possible that the third cell type stores a third member of the gastrin-CCK family of gut hormones.     

Distribution of gastrin and CCK cells in the rat gastrointestinal tract. Evidence for the occurrence of three distinct cell types storing COOH-terminal gastrin immunoreactivity.

Gastrin and cholecystokinin (CCH) cells of the rat gastrointestinal tract have been studied by immunocytochemistry and radioimmunoanalysis. With antisera directed against the COOH-terminal tetrapeptide sequence, which is common to gastrin and CCK, three distinct endocrine cell types are detected. One of the cell types predominates in the antrum, is scarce in the rest of the gut and corresponds to the gastrin cell. The second cell type is virtually confined to the duodenum and jejunum and corresponds to the CCK cell. The third cell type occurs disseminated in the small intestines, predominates in the ileum, and reacts with COOH-terminus-specific antisera only following diethylpyrocarbonate and not following formaldehyde fixation. It is possible that the third cell type stores a third member of the gastrin-CCK family of gut hormones.     

八肽胆囊收缩素在猪,大鼠和豚鼠肠道的免疫组织化学定位和分布

实验采用ＡＢＣ免疫组织化学方法研究了八肽胆囊收缩素样免疫反应物质在猪、大鼠和豚鼠肠道的定位与分布,并用相邻切片免疫双标记法观察了它与５－羟色胺的关系,结果表明,ＣＣＫ－８－ＩＲ细胞主要位于肠腺的底部,少数位于绒毛上皮。节段性分布上,在猪可见于从十二指肠到结肠全长的粘膜,大鼠和豚鼠ＣＣＫ－８－ＩＲ细胞则见于从十二指肠至回肠的粘膜,但均以十二指肠密度最高;免疫双标记法证实,在三种动物肠道中均有ＣＣＫ＝     

Immunohistochemical study of the distribution of endocrine cells in the gastrointestinal tract of the camel (Camelus bactrianus)

The regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the camel, Camelus bactrianus, were investigated using immunohistochemical methods. Ten types of immunoreactive (IR) endocrine cells were identified in this study. Among these cell types, only serotonin- and somatostatin-IR cells were detected in almost all regions of the gastrointestinal tract. Most of the cell types showed peak density in the pyloric gland region. The others showed restricted distribution: gastrin, cholecystokinin (CCK), motilin, bovine pancreatic polypeptide (BPP), and (gastric) substance P in the stomach; gastrin, CCK, BPP, gastric inhibitory polypeptide (GIP), glucagon, peptide tyrosine tyrosine (PYY) and substance P in the small intestine; and CCK, motilin, BPP, and PYY in the large intestine. Fundamentally the distribution pattern of endocrine cells in the gastrointestinal tract of the camel is similar to that of cattle. The distribution and frequency of endocrine cells in the glandular sac region are the same as those of the cardiac gland.     

Localization of xenin-immunoreactive cells in the duodenal mucosa of humans and various mammals.

Xenin is a 25-amino-acid peptide extractable from mammalian tissue. This peptide is biologically active. It stimulates exocrine pancreatic secretion and intestinal motility and inhibits gastric secretion of acid and food intake. Xenin circulates in the human plasma after meals. In this study, the cellular origin of xenin in the gastro-entero-pancreatic system of humans, Rhesus monkeys, and dogs was investigated by immunohistochemistry and immunoelectron microscopy. Sequence-specific antibodies against xenin detected specific endocrine cells in the duodenal and jejunal mucosa of all three species. These xenin-immunoreactive cells were distinct from enterochromaffin, somatostatin, motilin, cholecystokinin, neurotensin, and secretin cells, and comprised 8.8% of the chromogranin A-positive cells in the dog duodenum and 4.6% of the chromogranin A-positive cells in human duodenum. In all three species, co-localization of xenin was found with a subpopulation of gastric inhibitory polypeptide (GIP)-immunoreactive cells. Immunoelectron microscopy in the canine duodenal mucosa demonstrated accumulation of gold particles in round, homogeneous, and osmiophilic secretory granules with a closely adhering membrane of 187 +/- 19 nm diameter (mean +/- SEM). This cell type was found to be identical to the previously described canine GIP cell. Immunocytochemical expression of the peptide xenin in a subpopulation of chromogranin A-positive cells as well as the localization of xenin immunoreactivity in ultrastructurally characterized secretory granules permitted the identification of a novel endocrine cell type as the cellular source of circulating xenin.     

Comparative effects of galanin on isolated smooth muscle cells from ileum in five mammalian species.

Effect of galanin and CCK8 were studied on isolated smooth muscle cells obtained from pig, guinea-pig, rat, rabbit and dog ileum circular muscle layer. Galanin as well as CCK8 induced a concentration-dependent contraction of pig, rat, rabbit and guinea-pig ileum smooth muscle cells. Maximal contraction ranged between 23.7 +/- 1.9% and 26.1 +/- 3.1% decrease in cell length from control in the presence of both peptides. This maximal contraction was obtained at 1 nM galanin in pig, rat, rabbit, 1 nM CCK8 in rat, rabbit, guinea-pig, at 10 nM galanin in guinea-pig and 10 nM CCK8 in pig. Concentrations of galanin inducing a half maximal contraction (EC50) ranged between 8 pM and 80 pM in these species. In dog, CCK8 induced a concentration-dependent contraction of ileum smooth muscle cells, with a maximal contraction (24.5 +/- 2.3%) at 1nM and an EC50 of 50 pM while galanin inhibited cell contraction induced by CCK8. The CCK-induced contraction was abolished at 10 nM galanin and 10 nM VIP. Concentrations of galanin and VIP inducing a half-maximal relaxation of contracted cells were 2 pM and 3 pM respectively. It is concluded that galanin may induce cell contraction of pig, guinea-pig, rat and rabbit ileum circular muscle layer and cell relaxation of dog ileum by a direct myogenic effect.     

Morphological variation of immunoreactive cells positive to cholecystokinin 33 (10–20) and gastrin 34 (1–15) in human duodenum

Summary Human duodenal endocrine cells reactive with antibodies to cholecystokinin (CCK) 33 (10–20) and/or gastrin 34 (1–15) were studied by a combination of immunohistochemical and electron-microscopic methods. By immunohistochemistry, three types of endocrine cells were distinguished in human duodenal mucosa, i.e., those only positive for only CCK, those positive for both CCK and gastrin and those only positive for only gastrin. Ultrastructurally, the first cell type is characterized by many secretory granules with an eccentric dense core (mean diameter; 271+-74 nm). The second cell type, which was less frequent than the other two, has ultrastructural features that resemble type-I cells. The last cell type was composed of two types of cells containing small secretory granules identical to those of IG cells (mean diameter; 171+-31 nm) or large secretory granules indistinguishable from those of I cells (mean diameter; 286+-50 nm).     

Proliferative activity of gastric and duodenal endocrine cells in the rat

The replicative activity and migration of gastrin, somatostatin and serotonin cells in rat stomach and duodenum was studied using combined immunocytochemistry and autoradiography after 3H thymidine pulse-labeling. Our results show that a small proportion of gastrin, somatostatin and serotonin immunoreactive cells displays proliferative activity. The overall labeling index ranged from 1.3% for gastric endocrine cells to 3.2% for duodenal endocrine cells. In a pulse chase experiment, labeling indices of immunoreactive cells were estimated at several time intervals after 3H thymidine administration. Significant differences in labeling index were not found. Migration of 3H thymidine labeled endocrine cells towards the luminal surface was not found in the stomach nor in the duodenum. It is concluded that 1) these endocrine cells have replicating activity; 2) the replicative activity of endocrine cells is higher in the duodenum than in the stomach; 3) the various cell types do not show significant differences in replicating activity and 4) endocrine cells did not seem to migrate to the luminal surface of the mucosa along with the other epithelial cells.     

Leptin and CCK selectively activate vagal afferent neurons innervating the stomach and duodenum

The hormone leptin and the gut peptide CCK synergistically interact to enhance the process of satiation. Although this interaction may occur at several levels of the neuroaxis, our previous results indicate that leptin can specifically enhance the satiation effect of CCK by acting on subdiaphragmatic vagal afferent neurons. Because of this localized action, we hypothesized that a high proportion of vagal afferent neurons innervating the stomach or duodenum would be responsive to leptin and/or CCK. To test this hypothesis, we measured changes in cytosolic calcium levels induced by leptin and CCK in cultured nodose ganglion neurons labeled with a retrograde neuronal tracer injected into either the stomach or the duodenum. In the neurons labeled from the stomach, CCK activated 74% (39 of 53) compared with only 35% (34 of 97) of nonlabeled cells. Of the CCK-responsive neurons 60% (18 of 30) were capsaicin-sensitive. Leptin activated 42% (22 of 53) of the stomach innervating neurons compared with 26% of nonlabeled neurons. All of the leptin-sensitive neurons labeled from the stomach also responded to CCK. In the neurons labeled from the duodenum, CCK activated 71% (20 of 28). Of these CCK-responsive neurons 80% (12 of 15) were capsaicin sensitive. Leptin activated 46% (13 of 28) of these duodenal innervating neurons, of which 89% (8 of 9) were capsaicin-sensitive. Among neurons labeled from the duodenum 43% (12 of 28) were responsive to both leptin and CCK, compared with only 15% (15 of 97) of unlabeled neurons. Our results support the hypothesis that vagal afferent sensitivity to CCK and leptin is concentrated in neurons that innervate the stomach and duodenum. These specific visceral afferent populations are likely to comprise a substrate through which acute leptin/CCK interactions enhance satiation.     

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.     

Effects of cholecystokinin on acid formation in glands and cells isolated from rabbit and rat gastric mucosa

Isolated gastric glands and isolated cells prepared from rabbit and rat were studied to analyse the influence of cholecystokinin octapeptide (CCK 8) on histamine stimulated parietal cell acid formation as assessed by [14C]aminopyrine sequestered in acid tissue compartments. In rabbit gastric glands, CCK 8 evoked 32+/-6% (P<0. 01) inhibition of histamine stimulated acid formation, whereas in glands prepared from rat no inhibition was recorded. Instead, CCK 8 seemed to induce a variable increase of the histamine stimulation in rat gastric glands as the aminopyrine accumulation was increased by 110+/-46% (P<0.1). Further studies on cell preparations derived from rabbit gastric mucosa revealed dual properties of CCK 8, eliciting either inhibition or stimulation of the parietal cell depending on the presence of endocrine cells. The results show that paracrine communication may be effective in glandular preparations, but seems to vary depending on species.     

Changes in intestinal endocrine cells in the mouse after unilateral cervical vagotomy

The effect of right or left unilateral cervical vagotomy on the intestinal endocrine cells was studied in 23 mice at 2 and 8 weeks after operation, respectively. The results were compared with that from 10 sham operated mice. Various types of endocrine cells in duodenum and proximal colon were detected by immunohistochemistry and quantified by computerized image analysis. In mouse duodenum, chromogranin-, CCK/gastrin-, GIP- and somatostatin-cells were significantly decreased at 2 weeks after right vagotomy, but returned to the control levels at 8 weeks. Serotonin-cells were reduced at both 2 and 8 weeks after right vagotomy. The amount of the duodenal endocrine cells did not change after left vagotomy with the exception of secretin-cells, which were diminished at 8 weeks after both right and left vagotomy. In the proximal colon, chromogranin-cells were also decreased at 2 weeks after right vagotomy. Serotonin-cells were reduced at 8 weeks after left vagotomy but not right vagotomy. There was no significant difference between the unilaterally vagotomized and the sham operated mice with regard to PYY- and glucagon-cells. It was concluded that vagotomy affected the intestinal endocrine cells in mouse. The influence was more pronounced in the small intestine than the proximal colon. The right vagus nerves seemed to exert more effect on the intestinal endocrine cells than the left ones.     

Ultrastructural localization of secretin in endocrine cells of the dog duodenum by the immunogold technique

Summary Secretin has been localized by the immunogold technique in endocrine cells of the dog duodenum — previously described as K cells — characterized by secretory granules with double structure consisting of a secretin-containing osmiophilic core surrounded by an argyrophil halo. Granules resembling those of dog secretin cells were also found in some ultrastructurally characterized S cells of the cat, pig, rat and rabbit duodenum, thus confirming in these species the identification of S cells with secretin cells. Conversely, the cells previously described as S cells in the dog lacked secretin immunoreactivity.     

Spatiotemporal expression pattern of DsRedT3/CCK gene construct during postnatal development of myenteric plexus in transgenic mice

Cholecystokinin (CCK) is an early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. To determine the quantitative properties and the spatial distribution of the CCK-expressing myenteric neurones in early postnatal life, a transgenic mouse strain with a CCK promoter-driven red fluorescent protein (DsRedT3/CCK) was established. The cell-specific expression of DsRedT3/CCK was validated by in situ hybridization with a CCK antisense riboprobe and by in situ hybridization coupled with immunohistochemistry involving a monoclonal antibody to CCK. A gradual increase in the DsRedT3/CCK-expressing enteric neurones with clear regional differences was documented from birth until the suckling to weaning transition, in parallel with the period of rapid intestinal growth and functional maturation. To evaluate the proportion of myenteric neurones in which DsRedT3/CCK transgene expression was colocalized with the enteric neuronal marker peripherin, immunofluorescence techniques were applied. All DsRedT3/CCK neurones were peripherin-immunoreactive and the proportion of DsRedT3/CCK-expressing myenteric neurones in the duodenum was the highest after the third week of life, when the number of peripherin-immunoreactive myenteric neurones in this region had decreased. Nearly all of the DsRedT3/CCK-expressing neurones also expressed 5-hydroxytryptophan (5-HT). Thus, by utilizing a new transgenic mouse strain, we have demonstrated a small number of CCK-expressing myenteric neurones with a developmentally regulated spatiotemporal distribution. The coexistence of CCK and 5-HT in the majority of these neurones suggests their possible regulatory role in feeding at the suckling to weaning transition.     

Amylase secretion from isolated pure acinar cells

Isolation of pure acinar cells of the rat pancreas was achieved employing counterflow sedimentation filtration technique (CSFT). The preparation of purified acinar cells contained an occasional red blood cell (RBC, 200:1) with total absence of endocrine and duct cells. A significant stimulation of amylase secretion from isolated pure acinar cells was produced by octapeptide of cholecystokinin (CCK8) and insulin produced potentiation of the effect of CCK8. Synthetic glucagon inhibited basal and CCK8 stimulated amylase secretion. Non-synthetic purified glucagon stimulated amylase secretion and potentiated the effect of CCK8. Vasoactive intestinal polypeptide (VIP) did not stimulate amylase secretion but potentiated the effect of CCK8. No leakage of lactic dehydrogenase (LDH) was detected from the cells in any of the secretion studies. Thus a highly purified preparation of isolated pure acinar cells of rat pancreas could be obtained with excellent morphologic and functional integrity.     

Identification of enteroendocrine cells that express TRPA1 channels in the mouse intestine

TRPA1 is an ion channel that detects specific chemicals in food and also transduces mechanical, cold and chemical stimulation. Its presence in sensory nerve endings is well known and recent evidence indicates that it is expressed by some gastrointestinal enteroendocrine cells (EEC). The purpose of the present work is to identify and quantify EEC that express TRPA1 in the mouse gastrointestinal tract. Combined in situ hybridisation histochemistry for TRPA1 and immunofluorescence for EEC hormones was used. TRPA1 expressing EEC were common in the duodenum and jejunum, were rare in the distal small intestine and were absent from the stomach and large intestine. In the duodenum and jejunum, TRPA1 occurred in EEC that contained both cholecystokinin (CCK) and 5-hydroxytryptamine (5HT) and in a small number of cells expressing 5HT but not CCK. TRPA1 was absent from CCK cells that did not express 5HT and from EEC containing glucagon-like insulinotropic peptide. Thus TRPA1 is contained in very specific EEC populations. It is suggested that foods such as garlic and cinnamon that contain TRPA1 stimulants may aid digestion by facilitating the release of CCK.     

Activation of submucosal but not myenteric plexus of the gastrointestinal tract accompanies reduction of food intake by camostat

It has been shown in the rat that endogenous cholecystokinin (CCK), released in response to the non-nutrient trypsin inhibitor camostat, reduces food intake at meals and increases Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation) in the dorsal vagal complex (DVC) of the hindbrain but not the myenteric plexus of the duodenum and jejunum. Experiment 1: We examined Fos-LI in the myenteric and the submucosal plexuses of the gut in response to orogastric gavage of camostat in rats. As we reported previously, camostat failed to increase Fos-LI in the myenteric plexus. We show here that camostat increased Fos-LI in the submucosal plexus of the duodenum and jejunum. Camostat also increased Fos-LI in the DVC. Experiment 2: Pretreatment with devazepide, a specific CCK₁ receptor antagonist abolished camostat-induced Fos-LI in the submucosal plexus and the DVC. Experiment 3: Bilateral subdiaphragmatic vagotomy reduced camostat-induced Fos-LI in the submucosal plexus approximately 40% and abolished it in the DVC. Conclusions: Activation of the submucosal plexus by cholecystokinin at the CCK₁ receptor accompanies stimulation of the dorsal vagal complex of the hindbrain and inhibition of food intake. Unlike the submucosal plexus, activation of the myenteric plexus is not necessary for cholecystokinin's influence on the dorsal vagal complex and food intake. The lack of activation in the myenteric plexus after camostat stimulation, in contrast to nutrient releasers of CCK such as oleate, suggests that intestinal stimulants can either release different amounts of CCK or cause release of CCK from I cells with different molecular forms of CCK. This would suggest that CCK-8 is released by camostat and is not able to travel to the myenteric plexus while a more stable form of CCK such as CCK-58 can travel to this site that is further away from the I cell.