Gastric adenocarcinoma of fundic gland (chief cell predominant type) coexisting with well differentiated intestinal adenocarcinoma: A case report.

RATIONALE: Gastric adenocarcinoma of fundic gland (chief cell predominant type) (GA-FG-CCP) is a new, rare variant of gastric adenocarcinoma, which is characterized by mild nuclear atypia and specific immunohistochemical markers. PATIENT CONCERNS: An 84-year-old Chinese man was referred to our hospital for endoscopic resection of a gastric lesion. INTERVENTIONS: We performed endoscopic submucosal dissection, and successfully removed the lesion. DIAGNOSIS: Esophago gastroduodenoscopy showed a slightly elevated lesion with a diameter of 22 mm in the posterior wall of cardia. Magnifying endoscopy with narrow band imaging revealed an abnormal microsurface and microvessels on the tumor surface. Endoscopic ultrasonography revealed a hypoechoic mass located in the first layer. The pathological diagnosis of the biopsy specimens indicated that the tumor was high grade intraepithelial neoplasia. The pathological diagnosis differed between the superficial and deeper part of the lesion. The superficial part was composed of a tubular structure with prominent atypia and was diagnosed as well differentiated intestinal adenocarcinoma. The deeper part was composed of a well-differentiated tubular adenocarcinoma mimicking the fundic gland cells, mainly the chief cells. The tumor cells showed mild nuclear atypia and was positive for pepsinogen-I (PG-I) and mucin-6 (MUC6). This deeper part was diagnosed as GA-FG-CCP. OUTCOMES: The tumor was successfully removed. This patient had no discomfort during the follow-up period (10 months). LESSONS: We present a rare case of GA-FG-CCP coexisted with well-differentiated tubular adenocarcinoma. GA-FG-CCP exists in the deep mucosal layer and the muscularis mucosa, which could not be found under endoscopy, but could be discerned in pathology with mild nuclear atypia and special biomarkers.

Correlation Between the Number of Ghrelin-Secreting Cells in the Gastric Fundus and Excess Weight Loss after Sleeve Gastrectomy.

BACKGROUND: Weight loss after laparoscopic sleeve gastrectomy (LSG) has been mainly attributed to the restriction of gastric volume; however; other factors may contribute to weight loss after LSG. This study aimed to investigate the correlation between the number of ghrelin-secreting cells in the gastric fundus and excess weight loss (EWL) at 12 months after LSG. METHODS: The surface area of the gastric fundus was measured postoperatively in square centimeter. Histopathologic examination of the gastric fundus was made to estimate the number of ghrelin-secreting cells per square centimeter then was multiplied by the surface area of the fundus to calculate the total number of ghrelin-secreting cells in the fundus. The number of ghrelin-secreting cells was correlated with EWL and BMI at 12 months postoperatively. RESULTS: The present study included 39 patients of a mean age of 33.7 years. The mean %EWL at 12 months was 59.7 ± 12.7. The mean total number of ghrelin-producing cells in the gastric fundus was 26,228.4 ± 16,995.3. The total number of ghrelin-secreting cells had a weak positive correlation with BMI at 12 months (r = 0.2891, p = 0.07), and weak negative correlation with %EWL (r = - 0.1592, p = 0.33). CONCLUSION: There was a weak correlation between the total number of ghrelin-producing cells in the gastric fundus and plasma ghrelin levels with EWL after LSG.

Generation of human antral and fundic gastric organoids from pluripotent stem cells.

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.

Transdifferentiation of mucous neck cells into chief cells in fundic gastric glands shown by GNA lectin histochemistry.

The epithelium of the gastric mucosa and its glands in the corpus of rat stomach contains mucous surface cells (MSCs), parietal cells, mucous neck cells (MNCs), zymogenic or chief cells (ZCs), several types of enteroendocrine cells, and intermediate cells with characteristics between MNCs and ZCs also called transitional or prezymogenic cells (pre-ZCs). The aim of our work was to analyze the expression of Mannose (Man) in the rat gastric glands by means of Galanthus nivalis lectin (GNA) histochemistry to identify the differences between MNC, pre-ZCs and ZCs and to establish the relationships between these cells. Most of the cytoplasm of MNCs was negative for GNA histochemistry. Intensity of GNA labeling in the gastric gland showed a graduation from pre-ZCs (weak labeling) to ZCs (moderate labeling). Labeling of ZCs was stronger at the perinuclear and apical cytoplasm. In the last years, strong evidence has been reported supporting that ZCs differentiate from MNCs. Our work also supports the origin of ZCs from MNCs, because the GNA labeling graduation might be due to oligosaccharides which are not expressed in MNCs, start to express in pre-ZCs and are more abundant in ZCs, indicating that differentiation from MNCs to ZCs is a process in which glycans with Man moieties are synthesized.

Wnt/ß-catenin promotes gastric fundus specification in mice and humans.

Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.

Gastric adenocarcinoma of the fundic gland (chief cell-predominant type): A review of endoscopic and clinicopathological features.

Gastric adenocarcinoma of the fundic gland (chief cell-predominant type, GA-FG-CCP) is a rare variant of well-differentiated adenocarcinoma, and has been proposed to be a novel disease entity. GA-FG-CCP originates from the gastric mucosa of the fundic gland region without chronic gastritis or intestinal metaplasia. The majority of GA-FG-CCPs exhibit either a submucosal tumor-like superficial elevated shape or a flat shape on macroscopic examination. Narrow-band imaging with endoscopic magnification may reveal a regular or an irregular microvascular pattern, depending on the degree of tumor exposure to the mucosal surface. Pathological analysis of GA-FG-CCPs is characterized by a high frequency of submucosal invasion, rare occurrences of lymphatic and venous invasion, and low-grade malignancy. Detection of diffuse positivity for pepsinogen-I by immunohistochemistry is specific for GA-FG-CCP. Careful endoscopic examination and detailed pathological evaluation are essential for early and accurate diagnosis of GA-FG-CCP. Nearly all GA-FG-CCPs are treated by endoscopic resection due to their small tumor size and low risk of recurrence or metastasis.

Distribution and Ca(2+) signalling of fibroblast-like (PDGFR(+)) cells in the murine gastric fundus.

Platelet-derived growth factor receptor α positive (PDGFRα(+)) cells are suggested to mediate purinergic inputs in GI muscles, but the responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFRα(+) cells in murine gastric fundus, load cells with Ca(2+) indicators, and follow their activity via digital imaging. Immunolabelling demonstrated a high density of PDGFRα(+) cells in the fundus. Cells were isolated and purified by fluorescence-activated cell sorting (FACS) using endogenous expression of enhanced green fluorescent protein (eGFP) driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of purinergic P2Y1 receptors and SK3 K(+) channels in PDGFRα(+) cells. Ca(2+) imaging was used to characterize spontaneous Ca(2+) transients and responses to purines in PDGFRα(+) cells in situ. ATP, ADP, UTP and ß-NAD elicited robust Ca(2+) transients in PDGFRα(+) cells. Ca(2+) transients were also elicited by the P2Y1-specific agonist (N)-methanocarba-2MeSADP (MRS-2365), and inhibited by MRS-2500, a P2Y1-specific antagonist. Responses to ADP, MRS-2365 and ß-NAD were absent in PDGFRα(+) cells from P2ry1((-/-)) mice, but responses to ATP were retained. Purine-evoked Ca(2+) transients were mediated through Ca(2+) release mechanisms. Inhibitors of phospholipase C (U-73122), IP3 (2-APB), ryanodine receptors (Ryanodine) and SERCA pump (cyclopiazonic acid and thapsigargin) abolished Ca(2+) transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFRα(+) cells. Activation of Ca(2+) release is likely to be the signalling mechanism in PDGFRα(+) cells responsible for the transduction of purinergic enteric inhibitory input in gastric fundus muscles.

Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle.

Previous studies have identified differences in the expression of proteins that regulate myosin light chain phosphorylation and contraction in tonic and phasic smooth muscle. cGMP plays a critical role in smooth muscle relaxation and is important for optimal function of phasic and tonic smooth muscle. The intracellular cGMP levels are regulated by its hydrolysis via phosphodiesterase 5 (PDE5) and efflux via novel multidrug resistance protein 5 (MRP5). In the present study we tested the hypothesis that the differences in the phasic and tonic behavior of smooth muscles may be related to differences in mechanisms that terminate cGMP signaling. Expression of PDE5 and MRP5 was significantly (more than 2-fold) higher in fundus compared with antrum. The NO donor S-nitrosoglutathione (GSNO) caused an increase in PDE5 activity and intra- and extracellular cGMP levels in both fundus and antrum. Stimulation of PDE5 activity and increase in extracellular cGMP were significantly higher in fundus, whereas increase in intracellular cGMP was significantly higher in antrum. GSNO-induced increase in extracellular cGMP was blocked in dispersed cells by the cyclic nucleotide export blocker probenecid and in cultured muscle cells by depletion of ATP or suppression of MRP5 by siRNA, providing evidence that cGMP efflux was mediated by ATP-dependent export via MRP5. Consistent with the higher expression and activity levels of PDE5 and MRP5, GSNO-induced PKG activity and muscle relaxation were significantly lower in muscle cells from fundus compared with antrum. Thus higher expression of PDE5 and MRP5 in muscle cells from fundus correlates with tonic phenotype of muscle.

Interstitial cells of Cajal generate spontaneous transient depolarizations in the rat gastric fundus.

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were 15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 microM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 microM) also increased the amplitude of STDs and enhanced dV/dt(max). These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract.

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.

Functional role of bone morphogenetic protein-4 in isolated canine parietal cells.

Bone morphogenetic protein (BMP)-4 is an important regulator of cellular growth and differentiation. Expression of BMP-4 has been documented in the gastric mucosa. We reported that incubation of canine parietal cells with EGF for 72 h induced both parietal cell morphological transformation and inhibition of H(+)/K(+)-ATPase gene expression through MAPK-dependent mechanisms. We explored the role of BMP-4 in parietal cell maturation and differentiation. Moreover, we investigated if BMP-4 modulates the actions of EGF in parietal cells. H(+)/K(+)-ATPase gene expression was examined by Northern blots and quantitative RT-PCR. Acid production was assessed by measuring the uptake of [(14)C]aminopyrine. Parietal cell apoptosis was quantitated by Western blots with anti-cleaved caspase 3 antibodies and by counting the numbers of fragmented, propidium iodide-stained nuclei. MAPK activation and Smad1 phosphorylation were measured by Western blots with anti-phospho-MAPK and anti-phospho-Smad1 antibodies. Parietal cell morphology was examined by immunohistochemical staining of cells with anti-H(+)/K(+)-ATPase alpha-subunit antibodies. BMP-4 stimulated Smad1 phosphorylation and induced H(+)/K(+)-ATPase gene expression. BMP-4 attenuated EGF-mediated inhibition of H(+)/K(+)-ATPase gene expression and blocked EGF induction of both parietal cell morphological transformation and MAPK activation. Incubation of cells with BMP-4 enhanced histamine-stimulated [(14)C]aminopyrine uptake. BMP-4 had no effect on parietal cell apoptosis, whereas TGF-beta stimulated caspase-3 activation and nuclear fragmentation. In conclusion, BMP-4 promotes the induction and maintenance of a differentiated parietal cell phenotype. These findings may provide new clues for a better understanding of the mechanisms that regulate gastric epithelial cell growth and differentiation.

Immunocytochemical identification of interstitial cells of Cajal in the murine fundus using a live-labelling technique.

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells.

Gene expression profiling of gastrin target genes in parietal cells.

Previous studies demonstrated that mice with a null mutation in the gene encoding the hormone gastrin have impaired gastric acid secretion. Hence, the aim of this study was to evaluate changes in the acid-secreting parietal cell in gastrin-deficient (GAS-KO) mice. Analysis of several transcripts encoding parietal cell proteins involved in gastric acid secretion showed reduced abundance in the GAS-KO stomach, including H+,K+-ATPase alpha- and beta-subunits, KCNQ1 potassium channel, aquaporin-4 water channel, and creatine kinase B, which were reversed by gastrin infusion for 1 wk. Although mRNA and protein levels of LIM and SH3 domain-containing protein-1 (LASP-1) were not greatly changed in the mutant, there was a marked reduction in phosphorylation, consistent with its proposed role as a cAMP signal adaptor protein associated with acid secretion. A more comprehensive analysis of parietal cell gene expression in GAS-KO mice was performed using the Affymetrix U74AV2 chip with RNA from parietal cells purified by flow cytometry to >90%. Comparison of gene expression in GAS-KO and wild-type mice identified 47 transcripts that differed by greater than or equal to twofold, suggesting that gastrin affects parietal cell gene expression in a specific manner. The differentially expressed genes included several genes in signaling pathways, with a substantial number (20%) known to be target genes for Wnt and Myc.

Fasting plasma ghrelin concentrations 6 months after gastric bypass are not determined by weight loss or changes in insulinemia.

BACKGROUND: Ghrelin is a gastric peptide with potent orexigenic effects. Circulating ghrelin concentrations are increased in obese subjects, but increase after weight loss. However, in patients undergoing Roux-en-Y gastric bypass (RYGBP), a decrease in ghrelin levels has been reported. The effect of comparable weight loss induced by either adjustable gastric banding (AGB), RYGBP or conventional dietary treatment (Conv) on ghrelinemia was studied. METHODS: 24 matched obese male patients in whom similar weight loss had been achieved by either AGB (n=8), RYGBP (n=8) or Conv (n=8) were studied before and 6 months after treatment start. The independence of ghrelin concentrations from body mass index (BMI) and weight loss was further analyzed in a group of patients with total gastrectomy (TtGx, n=6). RESULTS: Comparable weight loss after 6 months exerted significantly different effects on plasma ghrelin concentrations, depending on the procedure applied (AGB: 424.6 +/- 32.8 pg/ml; RYGBP: 131.4 +/- 13.5; Conv: 457.3 +/- 18.7; P<0.001). Without significant differences in body weight and BMI, patients who had undergone the RYGBP exhibited a statistically significant decrease in fasting ghrelin concentrations, while the other two procedures (AGB and Conv) showed a weight loss-induced increase in ghrelin levels. Despite significant differences in BMI between RYGBP and TtGx patients after 6 months (31.9 +/- 2.2 vs 22.0 +/- 0.7 kg/m(2), respectively; P<0.05), both groups showed similar ghrelin concentrations. CONCLUSION: The reduction in circulating ghrelin concentrations in RYGBP patients after 6 months of surgery are not determined by an active weight loss or an improved insulin-sensitivity but rather depend on the surgically-induced bypass of the ghrelin-producing cell population of the fundus.

Characterization and species differences in gastric ghrelin cells from mice, rats and hamsters.

Ghrelin is a newly identified gastric peptide hormone that has various important functions, including growth-hormone release and appetite stimulation. Ghrelin-immunoreactive cells (ghrelin cells) are characterized by X-type endocrine cells in the rat stomach. In the present study, we analysed ghrelin cells in fundi of stomach from ICR mice and Syrian hamsters immunohistochemically, immunoelectron microscopically and morphometrically, and compared the results with those from Wistar rats. Immunohistochemistry revealed that ghrelin cells were sparsely distributed in the proper gastric glands in all species. The number of ghrelin cells per unit area in hamsters was significantly lower than that in rats. Immunoelectron microscopy detected ghrelin immunolabelling in granules in the X-type endocrine cells. However, the diameter of granules in the hamsters was significantly smaller than that in the mice and rats. Gastric ghrelin contents were determined by radioimmunoassay, and levels in the hamsters were significantly lower than those in mice and rats. The results from mice were identical to those from rats. In conclusion, gastric ghrelin cells in mice and hamsters are characterized by X-type endocrine cells, as has been observed in rats. However, the data indicated that gastric ghrelin production was lower in hamster than in mouse or rat.

Isolation of novel mouse genes that were differentially expressed in W/W(V) mouse fundus.

BACKGROUND: Using cDNA microarray analysis that displays a total of 8734 mouse genes, we have identified 21 known and novel transcripts, including mouse expressed sequence tags (ESTs) that were consistently up- or downregulated in the fundus of wild-type mice and W/W(V) mice, where intramuscular interstitial cells of Cajal (ICC; IC-IM) are lost. METHODS: By using these tags as part of the full-length mRNA sequence of the murine genes, we screened a mouse-brain cDNA library and the FASTA database (http://www.ebi.ac.uk/fasta33/). RESULTS: Three of the queries were identified as novel mouse genes, whereas six transcripts had their human counterparts that were known to encode functional proteins. Four transcripts, DRIM (downregulated in metastasis), SLP8 (tumor antigen), PTK7/CCK4 (receptor protein tyrosine kinase-like molecule-7/colon carcinoma kinase-4), and a novel gene AWMS2 were increased in W/W(V) mice. Expression of another five genes was decreased in W/W(V) mice: BB1 (overexpressed in bladder and breast carcinoma), HTATIP/CPLA2 (HIV-1 TAT interactive protein/cytosolic phospholipase A2), Tenascin-receptor like (Hexabrachion, Cytotactin, Neuronectin, Myotendinous antigen), and two novel transcripts: DRWMS1 and DRWMS2. Molecular profiling generated by cDNA microarray analysis from the fundus of W/W(V) mice and its complete list of full-length cDNAs will be shown on our web site (http://www.yamanashi.ac.jp/). CONCLUSIONS: Differential gene comparisons between control and mutant animals with losses in specific populations of ICCs will contribute significantly to our understanding of motility disorders associated with the loss of these cells and electrical slow waves in the gastrointestinal tract.

Regulation of amylin release from cultured rabbit gastric fundic mucosal cells.

BACKGROUND: Amylin (islet amyloid polypeptide) is a hormone with suggested roles in the regulation of glucose homeostasis, gastric motor and secretory function and gastroprotection. In the gastric mucosa amylin is found co-localised with somatostatin in D-cells. The factors regulating gastric amylin release are unknown. In this study we have investigated the regulation of amylin release from gastric mucosal cells in primary culture. Rabbit fundic mucosal cells enriched for D-cells by counterflow elutriation were cultured for 40 hours. Amylin and somatostatin release over 2 hours in response to agonists were assessed. RESULTS: Amylin release was significantly enhanced by activation of protein kinase C with phorbol-12-myristate-13-acetate, adenylate cyclase with forskolin and elevation of intracellular calcium with A23187. Cholecystokinin (CCK), epinephrine and glucagon-like peptide-1 (GLP-1) each stimulated amylin release in a dose-dependent manner. Maximal CCK-stimulated release was greater than either epinephrine or GLP-1, even when the effects of the latter two were enhanced by isobutylmethylxanthine. Stimulated amylin release was significantly inhibited by carbachol (by 51-59%) and octreotide (by 33-42%). Somatostatin release paralleled that of amylin. CONCLUSIONS: The cultured D-cell model provides a means of studying amylin release. Amylin secretion is stimulated by receptor-dependent and -independent activation of Ca2+/protein kinase C and adenylate cyclase pathways. Inhibition involves activation of muscarinic receptors and auto-regulation by somatostatin.

Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.

BACKGROUND: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel's diverticulum. METHODS: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form. RESULTS: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach. CONCLUSION: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.

Microarray comparison of normal and W/Wv mice in the gastric fundus indicates a supersensitive phenotype.

Interstitial cells of Cajal (ICC) have been identified in specific areas throughout the smooth musculature of the gastrointestinal (GI) tract. Located within the circular and longitudinal muscle layers of the gastric fundus lies a specific type of ICC, termed "intramuscular" ICC or IC-IM. The principal function of this cell type is to act as "mediators of excitatory and inhibitory enteric neurotransmission." The functional role of these cells has been investigated using W/W(v) mutant mice that specifically lack IC-IM, resulting in disrupted enteric neurotransmission. The aim of the present study was to investigate differential gene expression in W/W(v) mutant mice, from the tunica muscularis of the gastric fundus using a mouse cDNA microarray containing 1,081 known genes. Verification of the microarray data was attained using real-time "quantitative" PCR (qPCR). Of the 1,081 arrayed genes, 36 demonstrated differential expression by >2-fold in the W/W(v) mice. An agreement rate of 50% (7 of 14 tested) was obtained using qPCR. Of the seven confirmed changes in expression, several were indicative of a supersensitive phenotype, observed in denervation models. Expression of several putative neurotransmitter receptors including P2Y, the receptor for the inhibitory neurotransmitter ATP, was upregulated. The functional role of the P2Y receptor was also investigated using electrophysiological recordings. These results offer a new insight into the molecular changes that occur in W/W(v) fundic smooth muscle and may also provide novel information with regard to the importance of IC-IM in enteric neurotransmission.

Relaxation by vasoactive intestinal polypeptide in the gastric fundus of nitric oxide synthase-deficient mice.

In many gastrointestinal tissues nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) both play a role as inhibitory non-adrenergic non-cholinergic neurotransmitters. As the mode of interaction between NO and VIP remains controversial, the aim of this study was to investigate the interplay between NO and VIP in the mouse gastric fundus and to evaluate the nitric oxide synthase (NOS) isoform involved in VIP-induced relaxation by using inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) knockout mice. The influence of NOS inhibitors on the relaxant effect of VIP was determined in isolated smooth muscle cells and smooth muscle strips of wild-type and knockout mice. In isolated smooth muscle cells from wild-type, eNOS knockout and nNOS knockout mice, the relaxation induced by VIP (10(-9) M) was inhibited by approximately 70-95 % by both the non-selective NOS inhibitor N(G)-nitro-L-arginine (L-NA; 10(-4) M) and the selective inducible NOS inhibitor N-(3-(aminomethyl)-benzyl)acetamidine (1400W; 10(-6) M). In cells isolated from iNOS knockout mice, VIP still induced full relaxation but it was not influenced by L-NA or 1400W. In smooth muscle strips from wild-type and knockout mice, the concentration-dependent relaxation by VIP (10(-9) to 3 x 10(-7) M) was not influenced by L-NA or 1400W. These results suggest that the experimental method determines the influence of NOS inhibitors on the relaxant effect of VIP. iNOS, probably induced by the isolation procedure, might be involved in the relaxant effect of VIP in isolated smooth muscle cells but not in classic smooth muscle strips.