A cross-sectional study of nausea in functional abdominal pain: relation to mucosal mast cells and psychological functioning.

BACKGROUND: Nausea is a common symptom in youth with chronic abdominal pain. The aims of the current study were to assess: 1) the frequency of nausea in patients with functional dyspepsia (FD) and irritable bowel syndrome (IBS), respectively, as defined by Rome IV criteria; and, 2) relationships between nausea and mucosal inflammation as defined by antral and duodenal eosinophil and mast cell densities. A secondary aim was to assess relationships between nausea and other gastrointestinal symptoms, non-gastrointestinal somatic symptoms, and psychological dysfunction. METHODS: Records from patients with pain associated functional gastrointestinal disorders were retrospectively reviewed for gastrointestinal and somatic symptoms and anxiety, depression, and somatizations scores as assessed by the Behavior Assessment System for Children (BASC-2). In addition, previous gastric and mucosal biopsies were assessed for mast cell and eosinophil densities, respectively. RESULTS: 250 patients, ages 8 to 17 years, were assessed. Nausea was reported by 78% and was equally prevalent in those with FD alone, those with IBS alone, and those with both FD and IBS. Nausea was associated with increased mean (21.4 vs. 17.5) and peak (26.2 vs. 22.9) duodenal mast cell densities as compared those without nausea. Nausea was also associated with a wide variety of individual gastrointestinal symptoms, as well as headaches, fatigue, and dizziness. Lastly, nausea was associated with elevated self-report scores for anxiety (55.2 vs. 50.0), depression (50.2 vs. 46.1), and somatization (70.3 vs. 61.8). CONCLUSIONS: Nausea is common in children and adolescents with pain-associated FGIDs as defined by Rome IV and is not unique to either FD or IBS. Nausea is associated with increased mucosal mast cell density, non-gastrointestinal somatic symptoms, and psychologic dysfunction.

Notch signaling regulates gastric antral LGR5 stem cell function.

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5(+) antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5-GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5(+) stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi-colored reporter demonstrated that Notch-activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD-induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper-proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.

A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.

BACKGROUND AND AIMS: Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation. METHODS: Gastric glands were isolated from healthy human stomach tissue and grown in Matrigel containing media supplemented with various growth factors, developmental regulators and apoptosis inhibitors to generate long-lasting normal epithelial cell cultures. RESULTS: Culture conditions were developed which support the formation and quasi-indefinite growth of three dimensional (3D) spheroids derived from various sites of the human stomach. Spheroids could be differentiated to gastric organoids after withdrawal of Wnt3A and R-spondin1 from the medium. The 3D cultures exhibit typical morphological features of human stomach tissue. Transfer of sheared spheroids into 2D culture led to the formation of dense planar cultures of polarised epithelial cells serving as a suitable in vitro model of H. pylori infection. CONCLUSIONS: A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach.

Complex morphology of gastrin-releasing G-cells in the antral region of the mouse stomach.

Gastrin-releasing enteroendocrine cells (G-cells) are usually described as flask-shaped cells with a large base and a small apical pole, integrated in the epithelium lining the basal region of the antral invaginations in the stomach. By means of a transgenic mouse line in which the enhanced version of GFP is endogenously expressed under the control of a gastrin promoter, we have analyzed the spatial distribution and morphological features of G-cells. We found that G-cells were not only located at the basal region of the invagination but to a lesser extent also at the upper region. Visualization of the entire cellular morphology revealed that G-cells show complex morphologies. Basally located G-cells are roundish-shaped cells which project a prominent apical process towards the lumen and extend basal protrusions containing the hormone gastrin that were frequently found in close proximity to blood vessels and occasionally in the vicinity of nerve fibers. Inspection of G-cells in the upper region of antral invaginations disclosed a novel population of G-cells. These cells have a spindle-like contour and long apical and basal processes which extend vertically along the antral invagination, parallel to the lumen. This G-cell population seems to be in contact with a network of nerve fibers. While the functional role of these untypical G-cells is still elusive, the results of this study provide some useful indications to possible roles of these G-cells.

Effects of Exogenous Interleukin-9 on the Growth, Proliferation and Activity of Interstitial Cells of Cajal Cultured in vitro.

OBJECTIVES: To evaluate the role of exogenous interleukin-9 (IL-9) in the growth, proliferation and activity of interstitial cells of Cajal (ICCs) cultured in vitro, and to assess its role in maintaining the functions of ICCs. METHODS: ICCs of murine gastric antrum were isolated and cultured in vitro. ICCs were identified with c-Kit and ANO1 immunofluorescent antibodies. Both fluorescence microscope and confocal microscopy were used to observe the effects of IL-9 on the growth, proliferation and activity of ICCs in cultured in vitro. ICCs were loaded with fluorescence probe Fluo-3/AM and the fluorescence of intracellular calcium concentration ([Ca2+]i) was measured by confocal microscopy. The effects of exogenous IL-9 on the sulfated cholecystokinin octapeptide (CCK-8S)-evoked [Ca2+]i elevation were observed by confocal microscopy. RESULTS: Immunofluorescence results confirmed the successful separation and culture of ICCs in vitro. IL-9 in concentrations ranging from 0.02 to 1 µg/ml promoted the growth, proliferation and activity of ICCs in culture, and ICCs grew best with 0.5 mg/ml of IL-9. The presence of IL-9 could significantly increase the CCK-8S-evoked [Ca2+]i oscillation, which is probably caused by facilitating the maintenance of the functions of ICCs under suitable conditions for culture. CONCLUSION: IL-9 could promote the growth, proliferation and activity of ICCs, reinforce the CCK-8S-induced [Ca2+]i increment in ICCs, and facilitate the maintenance of the functions of ICCs under suitable culture condition.

CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis.

OBJECTIVE: Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. DESIGN: We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. RESULTS: Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5(neg or low) cell population but was distinct from typical antral Lgr5(high) stem cells. Treatment with progastrin interconverts Lgr5(neg or low) CCK2R+ cells into Lgr5(high) cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. CONCLUSIONS: CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum.

OBJECTIVE: The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. DESIGN: We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. RESULTS: The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. CONCLUSIONS: Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations.

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum.

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

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.

[Effect of banxia xiexin decoction on gastric antral interstitial cells of Cajal and stem cell factor in diabetic rats].

OBJECTIVE: To observe the effect of Banxia Xiexin Decoction (BXD) on expression of the interstitial cells of Cajal (ICCs) and stem cell factor (SCF) in the antrum of rats with diabetes mellitus (DM). METHODS: Totally sixty healthy male SD rats were randomly divided into the control group, the model group, the BXD group, and the Western medicine group (WM, treated by domperidone), 15 in each group. Diabetic rat models were established by a single intraperitoneal injection of streptozotocin (STZ, 55 mg/kg). Those in the BXD group were perfused with BXD at the daily dose of 5.4 g/kg. An equal volume of distilled water was given by gastrogavage to those in the WM group and the control group for 8 successive weeks. The body weight and blood glucose of all rats were detected, and the gastric residual rates were detected with semisolid nutrient paste by gastrogavage. The expression of positive ICCs and SCF were observed by immunohistochemical method and quantified image analyzer. RESULTS: Compared with the control group,the body weight reduced, blood glucose and gastric residual rates increased, and the mean optical density of positive ICCs and SCF significantly decreased in the model group (P < 0.05). Compared with the model group,symptoms such as polydipsia, polyphagia, polyuria were relieved, spirits improved, the body weight and mean optical densities of positive ICCs and SCF significantly increased (P < 0.05), and gastric residual rates significantly decreased in the BXD group and the WM group (P < 0.05). The blood glucose significantly decreased (P < 0.05) in the BXD group. The mean optical density of positive ICCs was higher in the BXD group than in the WM group (P < 0.05). CONCLUSIONS: BXD could promote the expression of positive ICCs and SCF. It could improve the gastric motility in DM rats by partially inverting abnormal changes of gastric antral ICCs and SCF.

[The role of gastric APUD system in progression of chronic Helicobacter gastritis].

The study included 60 patients with chronic Halicobacter gastritis (30 with chronic non-atrophic and 30 with atrophic Halicobacter gastritis (CNAHG and CAHG)). The control group was comprised of 15 practically healthy subjects. The aim of the work was to elucidate the role of Helicobacter infection, disturbances of regeneration, endothelin-1 and melatonin-secreting neuroendocrine cells of gastric antrum in progression of chronic Helicobacter gastritis (CHG). It was shown that CHG is due to H. pylori persistence and that patients with CNAHG undergo grade III microbial contamination while in CAHG patients atrophic changes are accompanied by metaplasia of gastric mucosa (GM) and inflammation of different severity. Patients with CNAHG has an increased number of melatonin-positive gastric cells and enhanced apoptotic activity of GM epitheliocytes. Patients with CAHG experience a reduction of melatonin-positive cells correlated with enhanced apoptotic activity of GM epitheliocytes. The number of endothelin-1 positive cells in patients with CNAHG and CAHG was similar to that in controls. Adequate eradication promoted normalization of the number of gastric endothelin-1 and melatonin-secreting neuroendocrine cells in patients with CHG. The apoptotic index reached the control value within 1 month after eradication in CNAHG patients but remained relatively high in CAHG patients.

Helicobacter pylori requires TlpD-driven chemotaxis to proliferate in the antrum.

Different disease outcomes of Helicobacter pylori infection correlate with distinct inflammation patterns. These different inflammatory distributions may be initiated by differences in bacterial localization. One H. pylori property known to affect murine stomach localization is chemotaxis, the ability to move in response to chemical cues. In this report, we used nonchemotactic mutants (Che(-)) to analyze whether chemotaxis is required for initial colonization of particular stomach regions or for subsequent growth therein. We found that H. pylori behaves differently in the corpus, antrum, and corpus-antrum transition zone subregions of the stomach. This outcome suggests that these regions contain unique chemotactic signals. In the corpus, H. pylori utilizes chemotaxis for initial localization but not for subsequent growth. In contrast, in the antrum and the corpus-antrum transition zone, chemotaxis does not help initial colonization but does promote subsequent proliferation. To determine which chemoreceptor is responsible for the corpus-antrum phenotypes, we infected mice with strains lacking each chemoreceptor. Strains lacking TlpA, TlpB, or TlpC displayed only modest deviations from the wild-type phenotype, while strains lacking TlpD resembled the Che(-) mutant in their antral colonization defect and fared even worse than the Che(-) mutant in the corpus. Additional analysis showed that inflammation is worse in the antrum than in the corpus in both wild-type and Che(-) mutant infections. These results suggest that chemotaxis, specifically, that controlled by TlpD, is necessary for H. pylori to survive or grow in the environment of increased inflammation in the antrum.

Conditional deletion of menin results in antral G cell hyperplasia and hypergastrinemia.

Antral gastrin is the hormone known to stimulate acid secretion and proliferation of the gastric corpus epithelium. Patients with mutations in the multiple endocrine neoplasia type 1 (MEN1) locus, which encodes the protein menin, develop pituitary hyperplasia, insulinomas, and gastrinomas in the duodenum. We previously hypothesized that loss of menin leads to derepression of the gastrin gene and hypergastrinemia. Indeed, we show that menin represses JunD induction of gastrin in vitro. Therefore, we examined whether conditional deletion of Men1 (Villin-Cre and Lgr5-EGFP-IRES-CreERT2), with subsequent loss of menin from the gastrointestinal epithelium, increases gastrin expression. We found that epithelium-specific deletion of Men1 using Villin-Cre increased plasma gastrin, antral G cell numbers, and gastrin expression in the antrum, but not the duodenum. Moreover, the mice were hypochlorhydric by 12 mo of age, and gastric somatostatin mRNA levels were reduced. However, duodenal mRNA levels of the cyclin-dependent kinase inhibitor p27(Kip1) were decreased, and cell proliferation determined by Ki67 staining was increased. About 11% of the menin-deficient mice developed antral tumors that were negative for gastrin; however, gastrinomas were not observed, even at 12 mo of age. No gastrinomas were observed with conditional deletion of Men1 in the Lgr5 stem cells 5 mo after Cre induction. In summary, epithelium-specific deletion of the Men1 locus resulted in hypergastrinemia due to antral G cell hyperplasia and a hyperproliferative epithelium, but no gastrinomas. This result suggests that additional mutations in gene targets other than the Men1 locus are required to produce gastrin-secreting tumors.

Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic {beta}-cells.

Hephaestin (Hp) is a membrane protein with ferroxidase activity that converts Fe(II) to Fe(III) during the absorption of nutritional iron in the gut. Using anti-peptide antibodies to predicted immunogenic regions of rodent Hp, previous immunocytochemical studies in rat, mouse, and human gut tissues localized Hp to the basolateral membranes of the duodenal enterocytes where the Hp was predicted to aid in the transfer of Fe(III) to transferrin in the blood. We used a recombinant soluble form of human Hp to obtain a high-titer polyclonal antibody to Hp. This antibody was used to identify the intracellular location of Hp in human gut tissue. Our immunocytochemical studies confirmed the previous localization of Hp in human enterocytes. However, we also localized Hp to the entire length of the gastrointestinal tract, the antral portion of the stomach, and to the enteric nervous system (both the myenteric and submucous plexi). Hp was also localized to human pancreatic beta-cells. In addition to its expression in the same cells as Hp, ferroportin was also localized to the ductal cells of the exocrine pancreas. The localization of the ferroxidase Hp to the neuronal plexi and the pancreatic beta cells suggests a role for the enzymatic function of Hp in the protection of these specialized cell types from oxidative damage.

Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection.

B7-H1 [programmed death-ligand-1 (PD-L1)] is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD-L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD-L1 expression, regulation and function during Helicobacter pylori infection. PD-L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co-culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD-L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon-γ or tumour necrosis factor-α. Moreover, PD-L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD-L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach.

Nutrient-sensing components of the mouse stomach and the gastric ghrelin cell.

BACKGROUND: The ability of the gut to detect nutrients is critical to the regulation of gut hormone secretion, food intake, and postprandial blood glucose control. Ingested nutrients are detected by specific gut chemosensors. However, knowledge of these chemosensors has primarily been derived from the intestine, while available information on gastric chemosensors is limited. This study aimed to investigate the nutrient-sensing repertoire of the mouse stomach with particular emphasis on ghrelin cells. METHODS: Quantitative RT-PCR was used to determine mRNA levels of nutrient chemosensors (protein: G protein-coupled receptor 93 [GPR93], calcium-sensing receptor [CaSR], metabotropic glutamate receptor type 4 [mGluR4]; fatty acids: CD36, FFAR2&4; sweet/umami taste: T1R3), taste transduction components (TRPM5, GNAT2&3), and ghrelin and ghrelin-processing enzymes (PC1/3, ghrelin O-acyltransferase [GOAT]) in the gastric corpus and antrum of adult male C57BL/6 mice. Immunohistochemistry was performed to assess protein expression of chemosensors (GPR93, T1R3, CD36, and FFAR4) and their co-localization with ghrelin. KEY RESULTS: Most nutrient chemosensors had higher mRNA levels in the antrum compared to the corpus, except for CD36, GNAT2, ghrelin, and GOAT. Similar regional distribution was observed at the protein level. At least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36. CONCLUSIONS AND INFERENCES: The cellular mechanisms for the detection of nutrients are expressed in a region-specific manner in the mouse stomach and gastric ghrelin cells. These gastric nutrient chemosensors may play a role modulating gastrointestinal responses, such as the inhibition of ghrelin secretion following food intake.

Biophysically based mathematical modeling of interstitial cells of Cajal slow wave activity generated from a discrete unitary potential basis.

Spontaneously rhythmic pacemaker activity produced by interstitial cells of Cajal (ICC) is the result of the entrainment of unitary potential depolarizations generated at intracellular sites termed pacemaker units. In this study, we present a mathematical modeling framework that quantitatively represents the transmembrane ion flows and intracellular Ca2+ dynamics from a single ICC operating over the physiological membrane potential range. The mathematical model presented here extends our recently developed biophysically based pacemaker unit modeling framework by including mechanisms necessary for coordinating unitary potential events, such as a T-Type Ca2+ current, Vm-dependent K+ currents, and global Ca2+ diffusion. Model simulations produce spontaneously rhythmic slow wave depolarizations with an amplitude of 65 mV at a frequency of 17.4 cpm. Our model predicts that activity at the spatial scale of the pacemaker unit is fundamental for ICC slow wave generation, and Ca2+ influx from activation of the T-Type Ca2+ current is required for unitary potential entrainment. These results suggest that intracellular Ca2+ levels, particularly in the region local to the mitochondria and endoplasmic reticulum, significantly influence pacing frequency and synchronization of pacemaker unit discharge. Moreover, numerical investigations show that our ICC model is capable of qualitatively replicating a wide range of experimental observations.

Somatostatin cell processes as pathways for paracrine secretion.

Somatostatin is produced by gastrointestinal endocrine cells that have long, nonluminal, cytoplasmic processes. Such processes terminate on other cell types, including gastrin-producing and hydrochloric acid-producing cells, whose functions are profoundly affected by somatostatin. The findings suggest that somatostatin cells control the functions of other cells through local release of the peptide by way of cytoplasmic processes. Also, certain other types of gastrointestinal endocrine cells have similar cytoplasmic prolongations, which may have analogous local (paracrine) regulatory functions.

Ca2+-activated K+ current in freshly isolated c-Kit positive cells in guinea-pig stomach.

This study was designed to isolate Ca2+-activated K+ current (I(KCa)) and elucidate its physiological significance in freshly isolated interstitial cells of Cajal (ICCs) of guinea-pig stomach. Single ICC was freshly isolated by enzymatically dissociating from myenteric border of gastric antrum free of circular muscles, and conventional whole-cell voltage clamp technique including immunohistochemical techniques were employed to characterize the cells: In myenteric border of gastric antrum, ICC-MY (ICCs from myenteric border) were detected by immunohistochemical reactivity, and single ICC-MY which has many branches was immunohistochemically c-Kit positive. Under K+-rich and 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid pipette solution, ICC produced spontaneous inward current (-256 +/- 92.2 pA). When step-depolarizing pulse from -80 to +80 mV was applied at holding potential (V(h)) of -80 mV, voltage-dependent outward currents were recorded with superimposed spontaneous transient outward currents (STOCs). Both STOCs and outward currents were reversibly affected by tetraethylammonium chloride (TEA) and iberiotoxin (IbTX); 2 mM TEA and 200 nM IbTX completely abolished STOCs and significantly inhibited outward K+ current over the whole potential range tested for current/voltage (I/V) relationship. In addition, TEA delayed repolarization phase of spontaneous inward current. The present results indicate the presence of I(KCa) in a single ICC, and it might be involved in regulation of repolarizing phase of spontaneous inward current in guinea-pig stomach.