Ingunn Bakke

A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control

Ghrelin is a 28 a.a. gastric peptide, recently identified as a natural ligand of the growth hormone secretagogue receptor (orphan receptor distinct from the receptor for growth hormone releasing hormone). In the present study, radioimmunoassay demonstrated ghrelin-like material in the rat oxyntic mucosa with moderate amounts also in antrum and duodenum. Small amounts were found in the distal intestines and pancreas. Northern blot analysis revealed abundant ghrelin mRNA in the oxyntic mucosa. Immunocytochemistry demonstrated ghrelin-immunoreactivity in endocrine-like cells in the oxyntic mucosa. Such cells occurred in low numbers also in the antrum and duodenum. The rat oxyntic mucosa is rich in endocrine (chromogranin A/pancreastatin-immunoreactive) cells, such as the histamine-rich ECL cells (65-75% of the endocrine cells), the A-like cells (20-25%) and the D cells (somatostatin cells) (10%). The ghrelin-immunoreactive (IR) cells contained pancreastatin but differed from ECL cells and D cells by being devoid of histamine-forming enzyme (ECL cell constituent) and somatostatin (D cell constituent). Hence, ghrelin seems to occur in the A-like cells. The ghrelin-IR cells in the antrum were distinct from the gastrin cells, the serotonin-containing enterochromaffin cells and the D cells. Conceivably, ghrelin cells in the antrum and distally in the intestines also belong to the A-like cell population. The concentration of ghrelin in the circulation was lowered by about 80% following the surgical removal of the acid-producing part of the stomach in line with the view that the oxyntic mucosa is the major source of ghrelin. The serum ghrelin concentration was higher in fasted rats than in fed rats; it was reduced upon re-feeding and seemed unaffected by 1-week treatment with the proton pump inhibitor omeprazole, resulting in elevated serum gastrin concentration. Infusion of gastrin-17 for 2 days failed to raise the serum ghrelin concentration. Omeprazole treatment for 10 weeks raised the level of HDC mRNA but not that of ghrelin mRNA or somatostatin mRNA in the oxyntic mucosa. Hence, unlike the ECL cells, ghrelin-containing A-like cells do not seem to operate under gastrin control.

Leptin Is Expressed in and Secreted from Primary Cultures of Human Osteoblasts and Promotes Bone Mineralization

The adipose hormone leptin and its receptor are important for regulation of food intake and energy metabolism. Leptin also is involved in the growth of different tissues. In this study, we show the expression of leptin in primary cultures of normal human osteoblasts (hOBs) as evidenced by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunocytochemistry. Release of leptin into the medium also was found. Leptin was not detected in commercially available hOBs (NHOst) or in three different human monoclonal osteosarcoma cell lines. Leptin expression was observed in OBs in the mineralization and/or the osteocyte transition period but not during the matrix maturation period. Furthermore, hOBs and osteosarcoma cell lines expressed the long signal‐transducing form of the leptin receptor (OB‐Rb) as shown by RT‐PCR. We observed no significant changes in leptin or OB‐Rb genes in hOBs after incubation with recombinant leptin, indicating no autoregulation of the leptin expression. Incubation of both hOBs entering the mineralization phase and osteosarcoma cell lines with recombinant leptin markedly increased the number of mineralized nodules as shown by alizarin S staining. These findings indicate that leptin may be of importance for osteoblastic cell growth and bone mineralization.

Long-Term Serotonin Administration Induces Heart Valve Disease in Rats

Background—The purpose of this study was to investigate whether rats dosed with serotonin develop changes similar to those seen in human carcinoid heart disease. Methods and Results—Ten Sprague-Dawley rats were given serotonin injections subcutaneously once daily for 3 months; controls were given saline. A long-lasting hyperserotoninemia with a >10-fold increase in both platelet-poor plasma and dialysate from the femoral muscles appeared. The animals developed clinical signs such as flushing and loose stools. After 3 months, 6 of 10 rats given serotonin had pathological echocardiographs. Two animals had a combination of aortic and pulmonary valve insufficiency, 1 had isolated aortic valve insufficiency, and 3 had isolated pulmonary valve insufficiency. Histopathological examination revealed shortened and thickened aortic cusps and carcinoidlike plaques characterized by a collection of myofibroblasts within an extracellular matrix of collagen ground substance. Immunostaining for Ki-67 demonstrated an increased number of proliferating subendocardial cells. In the control group, no pathological changes were seen. With the use of reverse-transcription polymerase chain reaction, normal rat aortic cusps were shown to express mRNA for serotonin receptors 5-HT1A, 5-HT2A, and 5-HT2B and the serotonin transporter 5-HTT. Conclusions—For the first time, long-term serotonin administration was performed in rats. Morphological and echocardiographic changes similar to those seen in human carcinoid heart disease developed. This study demonstrates that serotonin most likely is involved in the pathogenesis of carcinoid heart disease.

The CCK-2 Receptor is Located on the ECL Cell, But Not on the Parietal Cell

BACKGROUND The interrelationship between histamine and gastrin in the physiological regulation of gastric acid secretion is still a matter of dispute. CCK-2 receptors are located on enterochromaffin-like (ECL) cells in corpus mucosa and gastrin stimulates acid production by releasing histamine from the ECL cells, which in turn stimulates the parietal cells. Whether parietal cells also possess gastrin receptors of physiological significance is unclear. The aim of the present study was to localize the CCK-2 receptor cellularly and concomitantly demonstrate a gastrin receptor response (histamine release). METHODS Fluorescein labelled cholecystokinin-8 (Fluo-CCK-8) was added to the arterial infusion to totally isolated, vascularly perfused rat stomachs to a final concentration of 130 pmol L(-1) for 1 min, either alone or along with 520 nmol(-1) CCK-8 after 10-min pre-perfusion with CCK-8. Immediately after the Fluo-CCK-8 had reached the oxyntic mucosa, biopsies were taken and the binding sites were localized by double immunohistochemistry combined with the tyramide signal amplification (TSA) technique. Venous histamine was measured before and during stimulation. RESULTS Fluo-CCK-8 (130 pM) evoked histamine release, and binding sites were found in the basal part of corpus mucosa, co-localized with histidine decarbocylase (HDC) immunoreactive ECL cells. No binding of Fluo-CCK was found in the mid-glandular region of corpus, dominated by parietal cells. Binding of Fluo-CCK-8 was abolished by concomitant perfusion with excess CCK-8. CONCLUSION Fluo-CCK-8 given to isolated rat stomachs in a physiological concentration binds to CCK-2 receptors on ECL cells and causes histamine release, whereas no binding of Fluo-CCK-8 to parietal cells was found.Background: The interrelationship between histamine and gastrin in the physiological regulation of gastric acid secretion is still a matter of dispute. CCK-2 receptors are located on enterochromaffin-like (ECL) cells in corpus mucosa and gastrin stimulates acid production by releasing histamine from the ECL cells, which in turn stimulates the parietal cells. Whether parietal cells also possess gastrin receptors of physiological significance is unclear. The aim of the present study was to localize the CCK-2 receptor cellularly and concomitantly demonstrate a gastrin receptor response (histamine release). Methods: Fluorescein labelled cholecystokinin-8 (Fluo-CCK-8) was added to the arterial infusion to totally isolated, vascularly perfused rat stomachs to a final concentration of 130 pmol L -1 for 1 min, either alone or along with 520 nmol -1 CCK-8 after 10-min pre-perfusion with CCK-8. Immediately after the FluoCCK-8 had reached the oxyntic mucosa, biopsies were taken and the binding sites were localized by double immunohistochemistry combined with the tyramide signal amplification (TSA) technique. Venous histamine was measured before and during stimulation. Results: Fluo-CCK-8 (130 pM) evoked histamine release, and binding sites were found in the basal part of corpus mucosa, co-localized with histidine decarbocylase (HDC) immunoreactive ECL cells. No binding of Fluo-CCK was found in the midglandular region of corpus, dominated by parietal cells. Binding of Fluo-CCK-8 was abolished by concomitant perfusion with excess CCK-8. Conclusion: Fluo-CCK-8 given to isolated rat stomachs in a physiological concentration binds to CCK-2 receptors on ECL cells and causes histamine release, whereas no binding of Fluo-CCK-8 to parietal cells was found.

A new method for visualization of gut mucosal cells, describing the enterochromaffin cell in the rat gastrointestinal tract

Objective. Enterochromaffin (EC) cells in the gastrointestinal tract have an important function as regulators of secretion, motility and sensation. The EC cell has traditionally been described as bottle-shaped, with basally located stores of serotonin. Stimuli acting on the apical membrane trigger serotonin release, which in turn activates the subepithelial sensory nerve terminals. To better describe the appearance of EC cells, we developed a new method for visualization of mucosal cells. Material and methods. The stomach, small intestine and large intestine were excised from Sprague-Dawley rats and then fixed in formalin. The organs were everted and filled with pronase solution. Single cells and aggregates of formalin-fixed mucosal cells were collected by scraping the mucosa off the muscularis mucosa. EC cells were visualized by staining for immunoreactivity against serotonin. Results. EC cells with luminal extensions and very long (up to 80 µM) basally located axon-like extensions, sometimes connecting to neuron-like structures, were found. Other EC cells had no or only short and blunt basal extensions. Dividing serotonin-containing EC cells were also seen. Conclusions. These findings could make an important contribution towards furthering our understanding of EC cell function in gastrointestinal physiology. This new method can also readily be used to give better visualization of the morphology of other mucosal cells.

Hypergastrinemia in animals and man: causes and consequences

Gastrin was the second hormone to be postulated to exist (1) shortly after Bayliss & Starling (2) developed the concept of blood-borne chemical signal substances based upon their studies on the regulation of secretion of bicarbonate and water from the pancreas. Gastrin was postulated to be released from the antral mucosa by food and to reach the oxyntic mucosa to stimulate acid secretion (1). Many decades later, gastrin was purified, sequenced and synthesized (3) and shortly afterwards shown to be released by food (4) and to be a potent stimulator of acid secretion (5). The way gastrin stimulates the parietal cells to secrete acid has long been debated. It is now clear that gastrin stimulates acid secretion by inducing histamine release, which in turn stimulates acid secretion. This was shown as early as in the 1960s by Kahlson et al. (6), and later by us, using isolated vascularly perfused rat stomach (7) or isolated oxyntic mucosal cells (8). The cellular source of the histamine taking part in the regulation of acid secretion is nowadays undisputed as the ECL cell (9, 10). By comparing the concentration response curves for gastrin and histamine, we could show that the acid stimulatory effect of gastrin could be solely explained by the stimulation of histamine release (11), which we also had shown previously in vascularly perfused isolated rat stomach (12) or isolated oxyntic mucosal cells (8). Finally, by using a fluorescein labeled gastrin analogue, we could show that gastrin in physiological concentration only binds to the ECL cell and not to the parietal cell (13). In addition to stimulation of acid secretion, gastrin also has an undisputed positive trophic effect on the oxyntic mucosa (14). There is, however, a disproportionate trophic effect on the ECL cell (15). Whether there is a gastrin receptor on the exocrine mucosal stem cell in the oxyntic mucosa (16) or not (17), has not yet been settled. The general trophic effect of gastrin on the oxyntic mucosa may be indirectly mediated by ECL-cell-derived Reg protein (18). A gastrin receptor has also been localized to the C cells in the thyroid (19) and to tumors originating from this cell (19, 20), as well as to tumors originating from neuroendocrine cells in the lungs (21). The biological importance of these receptors has not been clarified, although pentagastrin-stimulated calcitonin release has previously been used in the diagnosis of medullary thyroid carcinoma (22). In the normal colon where gastrin does not influence mucosal growth (23), no gastrin receptor has been described. There have, however, been reports describing gastrin receptor in colonic carcinoma (24), but a relationship between gastrin and colonic carcinomas has not yet been confirmed, despite being the subject of debate for more than a decade. Furthermore, a physiological or pathophysiological role of progastrins including glycine-extended gastrin (25) has not been confirmed. The present review on hypergastrinemia will accordingly only focus on amidated gastrin and the principal target organ for gastrin, the oxyntic mucosa.

Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats

We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrins growth-promoting effect on the oxyntic mucosa.

Expression of CCL20 and Its Corresponding Receptor CCR6 Is Enhanced in Active Inflammatory Bowel Disease, and TLR3 Mediates CCL20 Expression in Colonic Epithelial Cells

Background The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production. Methods A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using in situ hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay. Results CCL20 and CCR6 mRNA abundances were increased during active inflammation (CCL20 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; CCR6 1.8 and 2.0, respectively). CCL20 and CCR6 mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and TLR3 silencing reduced CCL20 mRNA and protein levels. Conclusions The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.

Rat parietal cells express CCK2 receptor mRNA: gene expression analysis of single cells isolated by laser-assisted microdissection ☆

Gastrin plays a crucial role in maintaining a normal cellular composition and function of the oxyntic mucosa. It has been debated for decades whether parietal cells possess cholecystokinin-2 (CCK(2)) receptors and interact directly with gastrin. We investigated whether parietal cells express CCK(2) receptor mRNA by using new molecular biology techniques. Rat oxyntic mucosal cells were dispersed and enriched by elutriation, and single parietal and ECL cells were isolated from cell smears by means of laser microbeam microdissection and laser pressure catapulting. The mRNA from each single cell was isolated and subjected to one-step multiplex or conventional reverse transcription-polymerase chain reaction and subsequent nested PCR. Specific primers for the CCK(2) receptor were used in combination with primers for H,K-ATPase and histidine decarboxylase, specific markers for parietal and ECL cells, respectively. CCK(2) receptor mRNA was detected in 25% of the rat parietal cells and 40% of the ECL cells examined.

Microencapsulation of small intestinal neuroendocrine neoplasm cells for tumor model studies.

Basic cancer research is dependent on reliable in vitro and in vivo tumor models. The serotonin (5‐HT) producing small intestinal neuroendocrine tumor cell line KRJ‐1 has been used in in vitro proliferation and secretion studies, but its use in in vivo models has been hampered by problems related to the xeno‐barrier and tumor formation. This may be overcome by the encapsulation of tumor cells into alginate microspheres, which can function as bioreactors and protect against the host immune system. We used alginate encapsulation of KRJ‐1 cells to achieve long‐term functionality, growth and survival. Different conditions, including capsule size, variations in M/G content, gelling ions (Ca2+/Ba2+) and microcapsule core properties, and variations in KRJ‐1 cell condition (single cells/spheroids) were tested. Viability and cell growth was evaluated with MTT, and confocal laser scanner microscopy combined with LIVE/DEAD viability stains. 5‐HT secretion was measured to determine functionality. Under all conditions, single cell encapsulation proved unfavorable due to gradual cell death, while encapsulation of aggregates/spheroids resulted in surviving, functional bioreactors. The most ideal spheroids for encapsulation were 200–350 μm. Long‐term survival (>30 days) was seen with solid Ca2+/Ba2+ microbeads and hollow microcapsules. Basal 5‐HT secretion was increased (sixfold) after hollow microcapsule encapsulation, while Ca2+/Ba2+ microbeads was associated with normal basal secretion and responsiveness to cAMP/PKA activation. In conclusion, encapsulation of KRJ‐1 cells into hollow microcapsules produces a bioreactor with a high constitutively activate basal 5‐HT secretion, while Ca2+/Ba2+ microbeads provide a more stable bioreactor similar to non‐encapsulated cells. Alginate microspheres technology can thus be used to tailor different functional bioreactors for both in vitro and in vivo studies. (Cancer Sci 2012; 103: 1230–1237)