Search

Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption - due to a complete absence of enteroendocrine cells - followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic and, hence, may have had residual function in pancreas. We report 2 patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy but instead developed permanent IDDM during middle childhood ages. The variants showed no evidence of function in traditional promoter-based assays of NEUROG3 function and also failed to exhibit function in a variety of potentially novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that, unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression and, hence, suggest the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding β cell mass sufficient to maintain euglycemia until early childhood.

Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 μg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1β, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1β in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2 variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2 signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon.

Am J Physiol Gastrointest Liver Physiol 298: G45-G56, 2010. First published November 5, 2009; doi: 10.1152/ajpgi.00349.2009.--Peptide YY (PYY) antisecretory effect on intestinal epithelia is well established, whereas less is known about its actions to influence colonic motility in conscious animals. We characterized changes in basal function and stimulated colonic motor function induced by PYY-related peptides in conscious mice. [PYY.sub.3-36,] PYY, and neuropeptide Y (NPY) (8 nmol/ kg) injected intraperitoneally inhibited fecal pellet output (FPO) per hour during novel environment stress by 90%, 63%, and 57%, respectively, whereas the [Y.sub.1]-preferring agonists, [[Pro.sup.34]]pyY and [[Leu.sup.31,[Pro.sup.34]]]NpY, had no effect. Corticotrophin-releasing factor 2 receptor antagonist did not alter [PYY.sub.3-36] inhibitory action. PYY and PYY3-36 significantly reduced restraint-stimulated defecation, and PYY3-36 inhibited high-amplitude distal colonic contractions in restrained conscious mice for 1 h, by intraluminal pressure with the use of a microtransducer. PYY suppression of intraperitoneal 5-hydroxytryptophan induced FPO and diarrhea was blocked by the [Y.sub.2] antagonist, BIIE0246, injected intraperitoneally and mimicked by [PYY.sub.3-36,] but not [Leu31 ,Pro34]NpY. [PYY.sub.3-36] also inhibited bethanechol-stimulated FPO and diarrhea. [PYY.sub.3-36] inhibited basal FPO during nocturnal feeding period and light phase in fasted/refed mice for 2-3 h, whereas the reduction of food intake lasted for only 1 h. [PYY.sub.3-36] delayed gastric emptying after fasting-refeeding by 48% and distal colonic transit time by 104%, whereas [[Leu.sup.31,][Pro.sup.34]]NpY had no effect. In the proximal and distal colon, higher [Y.sub.2] mRNA expression was detected in the mucosa than in muscle layers, and [Y.sub.2] immunoreactivity was located in nerve terminals around myenteric neurons. These data established that PYY/[PYY.sub.3-36] potently inhibits basal and stress/serotonin/cholinergic-stimulated propulsive colonic motor function in conscious mice, likely via [Y.sub.2] receptors. [PYY.sub.3-36;] stress; serotonin; bethanechol

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the [CCK.sub.1] receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides. bioluminescence resonance energy transfer

In view of the importance of molecular sensing in the function of the gastrointestinal (GI) tract, we assessed whether signal transduction proteins that mediate taste signaling are expressed in cells of the human gut. Here, we demonstrated that the [alpha]-subunit of the taste-specific G protein gustducin (G[[alpha].sub.gust]) is expressed prominently in cells of the human colon that also contain chromogranin A, an established marker of endocrine cells. Double-labeling immunofluorescence and staining of serial sections demonstrated that G[[alpha].sub.gust] localized to enteroendocrine L cells that express peptide YY and glucagon-like peptide-1 in the human colonic mucosa. We also found expression of transcripts encoding human type 2 receptor (hT2R) family members, hT1R3, and G[[alpha].sub.gust] in the human colon and in the human intestinal endocrine cell lines (HuTu-80 and NCI-H716 cells). Stimulation of HuTu-80 or NCI-H716 cells with the bitter-tasting compound phenylthiocarbamide, which binds hT2R38, induced a rapid increase in the intracellular [Ca.sup.2+] concentration in these cells. The identification of G[[alpha].sub.gust] and chemosensory receptors that perceive chemical components of ingested substances, including drugs and toxins, in open enteroendocrine L cells has important implications for understanding molecular sensing in the human GI tract and for developing novel therapeutic compounds that modify the function of these receptors in the gut. type 2 receptor family; gastrointestinal peptides; peptide YY; glucagon-like peptide-1; chromogranin A; serotonin; phenylthiocarbamide

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the [alpha]-subunits of the G protein gustducin (G[[alpha].sub.gust]) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of [Ca.sup.2+] fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.i]) in a dose-and time-dependent manner. Chelating extracellular [Ca.sup.2+] with EGTA blocked the increase in [[[Ca.sup.2+]].sub.i] induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [[[Ca.sup.2+]].sub.i] induced by bombesin, but did not attenuate the [[[Ca.sup.2+]].sub.i] increase elicited by DB or PTC. These results indicate that [Ca.sup.2+] influx mediates the increase in [[[Ca.sup.2+]].sub.i] induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive [[Ca.sup.2+]] channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [[[Ca.sup.2+]].sub.i] elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [[[Ca.sup.2+]].sub.i] induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [[[Ca.sup.2+]].sub.i] and cholecystokinin release through [[Ca.sup.2+]] influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells. type 2 family taste receptors; gastrointestinal peptides; phospholipase C [[beta].sub.2]; [Ca.sup.2+] fluxes; enteroendocrine cells; cholecystokinin secretion

The genomic DNA from three unrelated patients with sparse enteroendocrine cells are screened for mutations of neurogenin-3 (NEUROG3) and then the ability of the observed mutations to alter NEUROG3 function is tested using in vitro and in vivo assays. Loss-of-function mutations in NEUROG3 causes a disorder characterized by malabsorptive diarrhea and a lack of intestinal enteroendocrine cells.

Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation of FcRn gene by the Sp family of transcription factors. Am J Physiol Gastrointest Liver Physiol 286: G922-G931, 2004; 10.1152/ ajpgi.00131.2003.--The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor (FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at -157/+ 135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines. passive immunity; development; ontogeny; immunoglobulin

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CC[K.sub.A] and CC[K.sub.B] receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-g for displacing the label from CC[K.sub.A] and CC[K.sub.B] receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity. cholecystokinin

Monoclonal and polyclonal antibodies were used to explore the localization of histidine decarboxylase (HDC) activity in the entero-chromaffin-like (ECL) cells of the oxyntic mucosa. Immunoreactive HDC-containing cells and HDC enzymatic activity demonstrated parallel decreases during fasting, parallel increases during refeeding and inhibition of the refeeding response by immunoneutralization or circulating gastrin. Immunostaining of histamine colocalized to the oxyntic ECL cell, but it did not change during fasting or refeeding.

The amplitude and stability of the membrane potential (Em) of purified rat peritoneal mast cells (PMCs) were analyzed to determine the effects of stem cell factors (SCF) on membrane integrity. Analysis of Em in purified PMCs from the rodent peritoneal lavage indicated the role of SCF in inducing Em instability which increased membrane capacitance. Furthermore, simultaneous recording of Em and real-time image analysis indicated the role of SCF in the maintenance and protection of purified PMCs in long-term culture.

An investigation of gastrin release in a murine model of hypergastrinemia induced by omeprazole, a drug for treating ulcers, has provided evidence to support that endogenous gastrin-releasing peptide (GRP) mediates gastrin secretion. It was shown that during hypergastrinemia, the inhibition of gastrin release activates a signal for the release of GRP, which, in turn, stimulates gastrin secretion.

Studies on the effect of anti-somatostatin antibody administration in suckling rats reveal that oral administration of monoclonal antibodies helps understand the role of various potential physiological regulators in the rats. Absorption of luminal monoclonal antibody is time-dependent and saturable. This is revealed by measuring serum murine immunoglobule G and the effects on serum and antral in gastrin concentration.

Background: Hypothalamic corticotropin‐releasing factor (CRF) receptor 1 (CRF1) plays a role in acute stress‐related stimulation of colonic motor function. Less is known on CRF1 signaling in the brainstem. Methods: We investigate CRF1 expression in the brainstem and the colonic response to 4th ventricle (4V) injection of CRF and urocortin (Ucn) 2 (3 µg/rat) in chronically cannulated male rats. Key results: Transcripts of CRF1 wild‐type 1a and splice variants 1c, 1e, 1f, 1o along with three novel variants 1a‐2 (desK‐110 in exon 5), 1p (‐exon 7), and 1q (exon 5 extension) were identified in the pons and medulla. The area postrema, nucleus tractus solitarius, dorsal motor nucleus of the vagus, locus coeruleus, and Barrington's nucleus isolated by laser capture microdissection expressed 1a, 1a‐2, and 1p but not 1q. Compared to 4V vehicle, 4V CRF induced fecal pellet output (FPO) and diarrhea that were blocked by the CRF antagonist, astressin‐B. CRF2 agonist, Ucn2 had no effect on basal or CRF‐induced FPO. CRF actions were correlated with the induction of c‐Fos immunoreactivity in myenteric neurons of the proximal and distal colon (pC, dC) and submucosal neurons of dC. c‐Fos immunoreactivity occurred in 39% and 37% of myenteric cholinergic and 7% and 58% of nitrergic neurons in the pC and dC, respectively. Conclusions & Inferences: CRF1a and its splice variants are expressed in brainstem nuclei, and activation of CRF1 signaling at the level of the brainstem stimulates colonic secretory‐motor function through activation of colonic enteric neurons. [ABSTRACT FROM AUTHOR]

Different actions of secretin and Gly-extended secretin predict secretin receptor subtypes. Am J Physiol Gastrointest Liver Physiol 280: G88-G94, 2001.--Only one secretin receptor has been cloned and its properties characterized in native and transfected cells. To test the hypothesis that stimulatory and inhibitory effects of secretin are mediated by different secretin receptor subtypes, pancreatic and gastric secretory responses to secretin and secretin-Gly were determined in rats. Pancreatic fluid secretion was increased equipotently by secretin and secretin-Gly, but secretin was markedly more potent for inhibition of basal and gastrin-induced acid secretion. In Chinese hamster ovary cells stably transfected with the rat secretin receptor, secretin and secretin-Gly equipotently displaced [sup.125]I-labeled secretin ([IC.sub.50] values 5.3 [+ or -] 0.5 and 6.4 [+ or -] 0.6 nM, respectively). Secretin, but not secretin-Gly, caused release of somatostatin from rat gastric mucosal D cells. Thus the equipotent actions of secretin and secretin-Gly on pancreatic secretion appear to result from equal binding and activation of the pancreatic secretin receptor. Conversely, secretin more potently inhibited gastric acid secretion in vivo, and only secretin re]eased somatostatin from D cells in vitro. These results support the existence of a secretin receptor subtype mediating inhibition of gastric acid secretion that is distinct from the previously characterized pancreatic secretin receptor. posttranslational processing; receptor specificity; D cells; somatostatin

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 µg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1β, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1β in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon. [ABSTRACT FROM AUTHOR]

Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF2) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF2 isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100μg/kg ip) over 24 h, and the effect of the CRF2 antagonist astresssin2-B (100 μg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF2b, the most common wild-type CRF2 isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF2b mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF2a, reportedly only found in the brain, and the novel splice variant CRF2a-3 were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF2a and an increase of CRF2a-3 in the GC 6 h postinjection. Astressin2-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF2 isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF2 signaling system may act to counteract the early gastric motor alterations to endotoxemia. [ABSTRACT FROM AUTHOR]

GLP-1 and its analog have been used in diabetes treatment; however, the direct alteration of gene expression profile in human islets induced by GLP-1 has not been reported. In present study, transcriptional gene expression in the liraglutide-treated human islets was analyzed with 12 human U133A chips including 23000 probe sets. The data compared between liraglutide and control groups showed a significant difference on glucose-induced insulin secretion, rather than viability. Microarray analysis identified 7000 genes expressed in human islets. Eighty genes were found to be modulated by liraglutide treatment. Furthermore, the products of these genes are proteins involved in binding capability, enzyme activity, transporter function, signal transduction, cell proliferation, apoptosis, and cell differentiation. Our data provides a set of information in the complex events, following the activation of the GLP-1 receptor in the islets of Langerhans. [ABSTRACT FROM AUTHOR]

Peptide YY (PYY) antisecretory effect on intestinal epithelia is well established, whereas less is known about its actions to influence colonic motility in conscious animals. We characterized changes in basal function and stimulated colonic motor function induced by PYY-related peptides in conscious mice. PYY3-36, PYY, and neuropeptide Y (NPY) (8 nmol/ kg) injected intraperitoneally inhibited fecal pellet output (FPO) per hour during novel environment stress by 90%, 63%, and 57%, respectively, whereas the Y -preferring agonists, [Pro34]PYY and [Leu31 ,Pro34]NPY, had no effect. Corticotrophin-releasing factor 2 receptor antagonist did not alter PYY3-36 inhibitory action. PYY and PYY3-36 significantly reduced restraint-stimulated defecation, and PYY3-36 inhibited high-amplitude distal colonic contractions in restrained conscious mice for 1 h, by intraluminal pressure with the use of a microtransducer. PYY suppression of intraperitoneal 5-hydroxytryptophan induced FPO and diarrhea was blocked by the Y2 antagonist, BIIE0246, injected intraperitoneally and mimicked by PYY3-36, but not [Leu31 ,Pro34]NPY. PYY3-36 also inhibited bethanechol-stimulated FPO and diarrhea. PYY3-36 inhibited basal FPO during nocturnal feeding period and light phase in fasted/refed mice for 2-3 h, whereas the reduction of food intake lasted for only 1 h. PYY3-36 delayed gastric emptying after fasting-refeeding by 48% and distal colonic transit time by 104%, whereas [Leu31,Pro34]NPY had no effect. In the proximal and distal colon, higher Y2 mRNA expression was detected in the mucosa than in muscle layers, and Y2 immunoreactivity was located in nerve terminals around myenteric neurons. These data established that PYY/PYY3-36 potently inhibits basal and stress/serotonin/cholinergic-stimulated propulsive colonic motor function in conscious mice, likely via Y2 receptors. [ABSTRACT FROM AUTHOR]

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the α-subunits of the G protein gustducin (Gαgust) in the rodent gastrointestinal (GI) tract and in G1 endocrine cells. In this study, we characterized mechanisms of Ca2+ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca2+] ([Ca2+]i) in a dose- and time-dependent manner. Chelating extracellular Ca2+ with EGTA blocked the increase in [Ca2+]i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca2+]i induced by bombesin, but did not attenuate the [Ca2+]i increase elicited by DB or PTC. These results indicate that Ca2+ influx mediates the increase in [Ca2+]i induced by DB and PTC in STC-l cells. Preincubation with the L-type voltage-sensitive Ca2+ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 mm inhibited the increase in [Ca2+]i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca2+]i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca2+]i and cholecystokinin release through Ca2+ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells. [ABSTRACT FROM AUTHOR]

Background: Cytotoxin-associated gene A (cagA) has been implicated as a potential pathogenic marker for Helicobacter pylori-induced severe gastroduodenal diseases. Although the prevalence of cagA-positive strains has been reported in patient populations from developed countries, only limited information from developing countries is available. Methods: Polymerase chain reaction (PCR) in combination with immunoblot analysis was used to determine the prevalence of cagA and its adjacent cagE genes and to evaluate the expression of CagA protein in 55 H. pylori clinical isolates from China. Results: The expected PCR products derived from H. pylori cagA and cagE genes were identified in all Chinese H. pylori clinical isolates. Similarly, the CagA protein was detected in all 40 isolates tested. Conclusions: These results demonstrated that the presence of the cagA gene correlated well with expression of the CagA protein in all surveyed Chinese H. pylori isolates and that infection with cagA-positive H. pylori strains is highly common in China and independent of clinical presentation. [ABSTRACT FROM AUTHOR]