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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.

The aim of this study is to investigate the efficacy of combining a histone deacetylase inhibitor (LBH589) and a breast cancer stem cells (BCSC)-targeting agent (salinomycin) as a novel combination therapy for triple-negative breast cancer (TNBC). We performed in vitro studies using the TNBC cell lines to examine the combined effect. We used the mammosphere and ALDEFLUOR assays to estimate BCSC self-renewal capacity and distribution of BCSCs, respectively. Synergistic analysis was performed using CalcuSyn software. For in vivo studies, aldehyde dehydrogenase 1 ALDH1-positive cells were injected into non-obese diabetic/severe combined immunodeficiency gamma (NSG) mice. After tumor formation, mice were treated with LBH589, salinomycin, or in combination. In a second mouse model, HCC1937 cells were first treated with each treatment and then injected into NSG mice. For mechanistic analysis, immunohistochemistry and Western blot analysis were performed using cell and tumor samples. HCC1937 cells displayed BCSC properties including self-renewal capacity, an ALDH1-positive cell population, and the ability to form tumors. Treatment of HCC1937 cells with LBH589 and salinomycin had a potent synergistic effect inhibiting TNBC cell proliferation, ALDH1-positive cells, and mammosphere growth. In xenograft mouse models treated with LBH589 and salinomycin, the drug combination effectively and synergistically inhibited tumor growth of ALDH1-positive cells. The drug combination exerted its effects by inducing apoptosis, arresting the cell cycle, and regulating epithelial-mesenchymal transition (EMT). Combination of LBH589 and salinomycin has a synergistic inhibitory effect on TNBC BCSCs by inducing apoptosis, arresting the cell cycle, and regulating EMT; with no apparent associated severe toxicity. This drug combination could therefore offer a new targeted therapeutic strategy for TNBC and warrants further clinical study in patients with TNBC.

Background & Aims: Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency., Methods: We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins., Results: We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears., Conclusions: In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Brain corticotropin-releasing factor (CRF) acting on CRF receptor type 1 (CRF(1)) is a main signaling pathway in the stress response. CRF is also produced in a variety of peripheral sites and acts locally as a proinflammatory mediator. We investigated CRF(1) mRNA expression in the human gastrointestinal tract, and localized CRF(1) immunoreactive cells in the colonic mucosa of healthy subjects and patients with ulcerative colitis (UC). In 4 male healthy subjects (24-29 years), CRF(1) transcript was detected by RT-PCR throughout the gastrointestinal tract with the highest levels in the ileum and rectum and the lowest level in the colon. Immunohistochemistry on whole thickness sigmoid colon sections showed that CRF(1) was localized in the lamina propria and epithelial cells and enteric neurons. In sigmoid colonic biopsies, immunohistochemically double-labeled cells with CRF(1) and CD163, a marker for macrophages, represent 79% of total CRF(1) immunoreactive (IR) cells in healthy subjects. In 10 UC patients, the total number of CRF(1) IR cells and CRF(1)/CD163 double-labeled macrophages was increased by 4.2 and 4.0 folds respectively compared to healthy subjects. These findings indicate that CRF(1) is distributed throughout the GI tract of healthy human subjects. The increase of CRF(1) IR cells prominently in macrophages of the sigmoid colonic mucosa of UC patients provides anatomical support for a role of CRF(1) signaling in modulating the immune-inflammatory process of UC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) 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 CRF(2) 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, 100 μg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 μg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) 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, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF(2) isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF(2) signaling system may act to counteract the early gastric motor alterations to endotoxemia.

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.

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.

Mice and rats are widely used in stress-related behavioral studies while little is known about the distribution of the stress hormone, corticotropin-releasing factor (CRF) in the mouse brain. We developed and characterized a novel rat/mouse CRF polyclonal antibody (CURE ab 200101) that was used to detect and compare the brain distributions of CRF immunoreactivity in naïve and colchicine-treated rats and mice. We also assessed whether the visceral stressor of abdominal surgery activated brain CRF neurons using double labeling of Fos/CRF in naïve rats. CRF-ir neurons were visualized in the cortex, bed nucleus of the stria terminalis, central amygdala, hypothalamic paraventricular nucleus (PVN), Barrington's nucleus and dorsolateral tegmental area in naïve rats. CRF-immunoreactive (ir) neurons in the mouse brain were detected only after colchicine. The pattern shows fundamental similarity compared to the colchicine-treated rat brain, however, there were differences with a lesser distribution in both areas and density except in the lateral septum and external subnucleus of the lateral parabrachial nucleus which contained more CRF-ir neurons in mice, and CRF-ir neurons in the dorsal motor nucleus of the vagus were found only in mice. Abdominal surgery in naïve rats induced Fos-ir in 30% of total CRF-ir neurons in the PVN compared with control (anesthesia alone) while Fos was not co-localized with CRF in other brain nuclei. These data indicate that CRF-ir distribution in the brain displays similarity as well as distinct features in mice compared to rats that may underlie some differential stress responses. Abdominal surgery activates CRF-ir neurons selectively in the PVN of rats without colchicine treatment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background & Aims: Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress., Methods: We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2)., Results: In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2)., Conclusions: In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

CRH and 5-hydroxytryptamine (5-HT) are expressed in human colonic enterochromaffin (EC) cells, but their interactions at the cellular level remain largely unknown. The mechanistic and functional relationship between CRH and 5-HT systems in EC cells was investigated in a human carcinoid cloned BON cell line (BON-1N), widely used as an in vitro model of EC cell function. First, we identified multiple CRH(1) splice variants, including CRH(1a), CRH(1c), CRH(1f), and a novel form lacking exon 4, designated here as CRH(1i), in the BON-1N cells. The expression of CRH(1i) was also confirmed in human brain cortex, pituitary gland, and ileum. Immunocytochemistry and immunoblot analysis confirmed that BON-1N cells were CRH(1) and 5-HT positive. CRH, urocortin (Ucn)-1, and cortagine, a selective CRH(1) agonist, all increased intracellular cAMP, and this concentration-dependent response was inhibited by CRH(1)-selective antagonist NBI-35965. CRH and Ucn-1, but not Ucn-2, stimulated significant ERK1/2 phosphorylation. In transfected human embryonic kidney-293 cells, CRH(1i) isoforms produced a significant increase in pERK1/2 in response to CRH(1) agonists that was sensitive to NBI-35965. CRH and Ucn-1 stimulated 5-HT release that reached a maximal increase of 3.3- and 4-fold at 10(-8) m over the basal level, respectively. In addition, exposure to CRH for 24-h up-regulated tryptophan hydroxylase-1 mRNA levels in the BON-1N cells. These findings define the expression of EC cell-specific CRH(1) isoforms and activation of CRH(1)-dependent pathways leading to 5-HT release and synthesis; thus, providing functional evidence of a link exists between CRH and 5-HT systems, which have implications in stress-induced CRH(1) and 5-HT-mediated stimulation of lower intestinal function.

Little is known about CRF expression and regulation in the rat colon compared to the brain. We investigated CRF gene expression, cellular location, and regulation by endotoxin and corticosterone in the male rat colon at 6h after intraperitoneal (ip) injection. CRF mRNA level, detected by reverse transcription-polymerase chain reaction (RT-PCR) was 1.3-fold higher in the distal than proximal colon and 3.4-fold higher in the proximal colonic submucosa plus muscle layers than in mucosa. CRF immunoreactivity was located in the epithelia, lamina propria and crypts, and co-localized with tryptophan hydroxylase, a marker for enterochromaffin (EC) cells, and in enteric neurons. Lipopolysaccharide (LPS, 100 microg/kg, ip) increased defecation by 2.9-fold and upregulated CRF mRNA by 2.5-fold in the proximal and 1.1-fold in the distal colon while there was no change induced by corticosterone as monitored by quantitative PCR. LPS-induced increased CRF mRNA expression occurred in the submucosa plus muscle layers (1.5-fold) and the mucosa of proximal colon (0.9-fold). LPS increased significantly CRF immunoreactivity in the submucosal and myenteric plexuses of proximal and distal colon compared to saline groups. These results indicate that in rats, CRF is expressed in both proximal and distal colon and more prominently in enteric neurons of the submucosa plus muscle layers and subject to upregulation at the gene and protein levels by LPS through corticosteroid independent pathways. These data suggests that colonic CRF may be part of the local effector limb of the CRF(1) receptor mediated colonic alterations induced by acute stress., ((c) 2009 Elsevier Inc. All rights reserved.)

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(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(1)-preferring agonists, [Pro(34)]PYY and [Leu(31),Pro(34)]NPY, had no effect. Corticotrophin-releasing factor 2 receptor antagonist did not alter PYY(3-36) inhibitory action. PYY and PYY(3-36) significantly reduced restraint-stimulated defecation, and PYY(3-36) 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(2) antagonist, BIIE0246, injected intraperitoneally and mimicked by PYY(3-36), but not [Leu(31),Pro(34)]NPY. PYY(3-36) also inhibited bethanechol-stimulated FPO and diarrhea. PYY(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(3-36) delayed gastric emptying after fasting-refeeding by 48% and distal colonic transit time by 104%, whereas [Leu(31),Pro(34)]NPY had no effect. In the proximal and distal colon, higher Y(2) mRNA expression was detected in the mucosa than in muscle layers, and Y(2) immunoreactivity was located in nerve terminals around myenteric neurons. These data established that PYY/PYY(3-36) potently inhibits basal and stress/serotonin/cholinergic-stimulated propulsive colonic motor function in conscious mice, likely via Y(2) receptors.

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 CCK1 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.

Matrix metalloproteinases (MMPs) are a family of endopeptidases that collectively are capable to degrading all components of the extracellular matrix (ECM) and they have been implicated in several aspects of tumor progression, such as invasion through basement membrane (BM) and insterstitial matrices, angiogenesis and tumor cell growth. In particular, MMP-2 and MMP-9 have been associated with the ability of tumor cells to metastasize due to their capacity to degrade type IV collagen (Col-IV), the main component of BM, and to their elevated expression in malignant tumors. However, nothing is known about the regulation of MMP-9 secretion and expression in breast cancer cells stimulated with Col-IV. Our results demonstrate that stimulation of MCF-7 cells with Col-IV promoted the secretion of MMP-9, as revealed by gelatin zymography and Western blotting using specific antibodies that recognized MMP-9. In addition, inhibition of Src and FAK kinase activity prevented MMP-9 secretion. In contrast, MMP-9 expression was not up-regulated by treatment with Col-IV. These results demonstrate that Col-IV regulates the secretion of MMP-9 via a Src and FAK dependent pathway in MCF-7 cells.

Intraperitoneal (i.p.) corticotropin releasing factor (CRF) induced a CRF(1) receptor-dependent stimulation of myenteric neurons and motility in the rat proximal colon. We characterize the colonic enteric nervous system response to CRF in conscious rats. Laser capture microdissection combined with reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry in longitudinal muscle myenteric plexus whole-mount colonic preparations revealed CRF(1) receptor expression in myenteric neurons. CRF (i.p., 10 microg kg(-1)) induced Fos immunoreactivity (IR) (cells per ganglion) selectively in myenteric plexus of proximal (18.3 +/- 2.4 vs vehicle: 0.0 +/- 0.0) and distal colon (16.8 +/- 1.2 vs vehicle: 0.0 +/- 0.0), but not in that of gastric corpus, antrum, duodenum, jejunum and ileum. The selective CRF(1) agonist, stressin(1)-A (i.p., 10 microg kg(-1)) also induced Fos IR in myenteric but not in submucosal plexus of the proximal and distal colon. Fos IR induced by CRF was located in 55 +/- 1.9% and 53 +/- 5.1% of CRF(1) receptor-IR myenteric neurons and in 44 +/- 2.8% and 40 +/- 3.9% of cholinergic neurons with Dogiel type I morphology, and in 20 +/- 1.6% and 80 +/- 3.3% of nitrergic neurons in proximal and distal colon respectively. CRF and stressin(1)-A elicit defecation and diarrhoea. These data support that one mechanism through which peripherally injected CRF ligands stimulate colonic function involves a direct action on colonic cholinergic and nitrergic myenteric neurons expressing CRF(1) receptor.

The rat esophagus shares some cellular features with skin squamous epithelium and striated muscle that express high levels of corticotropin-releasing factor type 2 (CRF2) receptors or their cognate ligand urocortin (Ucn) 1, 2, and 3. We investigated the expression and cell signaling of CRF2 receptors and ligands in the rat esophagus and lower esophageal sphincter (LES) by RT-PCR and quantitative PCR in normal and corticosterone-treated whole esophageal tissue, laser capture microdissected layers, and isolated esophageal cells. The expression of CRF2 receptor protein and intracellular cAMP and ERK1/2 responses to CRF agonists and CRF2 antagonist were determined in cultured esophageal cells and HEK-293 cells transfected with CRF2b receptors. CRF2 was abundantly expressed in the mucosa and longitudinal muscle layers of the esophagus and LES, whereas CRF1 expression was scarce. CRF2b wild-type transcript was predominantly expressed in the esophagus, and in addition, several new CRF2 splice variants including six CRF2a isoforms were identified. Expression of Ucn 1, Ucn 2, and to a smaller extent Ucn 3, but not CRF mRNA, was detected in the esophagus and LES. Ucn 1 and Ucn 2 stimulated dose-dependent cAMP production and ERK1/2 phosphorylation in the esophageal cells, whereas CRF and CRF1 agonist, cortagine, had less potent effects. In addition, Ucn 2-stimulated cAMP and ERK responses were blocked by the CRF2 antagonist, astressin2-B. These data established the presence of a prominent CRF2 signaling system in the esophagus and LES-encompassing multiple CRF2 receptor variants and Ucn, suggesting a functional role in secretomotor activity and epithelial and muscle cell proliferation.

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 (Galpha(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 Galpha(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 Galpha(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 Ca2+ concentration in these cells. The identification of Galpha(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.

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the alpha-subunits of the G protein gustducin (Galpha(gust)) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca(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(2+)] ([Ca(2+)](i)) in a dose- and time-dependent manner. Chelating extracellular Ca(2+) with EGTA blocked the increase in [Ca(2+)](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(2+)](i) induced by bombesin, but did not attenuate the [Ca(2+)](i) increase elicited by DB or PTC. These results indicate that Ca(2+) influx mediates the increase in [Ca(2+)](i) induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive Ca(2+) channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [Ca(2+)](i) elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca(2+)](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(2+)](i) and cholecystokinin release through Ca(2+) influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells.

Background: The efficacy of cardiopulmonary resuscitation (CPR) is vital for saving lives of victims with sudden cardiac arrest. In 1960, Kuowenhoven and colleagues proposed the method that has become standard for CPR. Despite vast input of resources for public education and training of this procedure, its success rate outside hospitals remains poor to dismal. During CPR, restoration of respiration is as important as circulation. But opening the airway and giving effective mouth-to-mouth respiration is difficult for lay people to learn. Furthermore, most bystanders are reluctant to do mouth-to-mouth respiration because of the risk of infection. Therefore, the general population needs a more simplified CPR method for outpatients. The practice of CPR in the prone position, first proposed by McNeil in 1989, has not been adopted, despite the fact that it meets the desirable requirements of ideal resuscitation: simultaneous restoration of circulation and respiration with a very simple maneuver., Methods: Part 1 (circulation test): Eleven patients who expired in the intensive care unit (ICU), with arterial lines attached, received standard pre-cordial cardiac massage, and the generated blood pressure (BP) was recorded. They were then turned to the prone position, with the head turned to one side. We compressed the patient's thoracic spine with the same force used in standard CPR (rhythm of approximately 60 per minute each time when the back bounces back), and the BP was also recorded. Part 2 (ventilation test): Ten healthy volunteers (5 doctors and 5 nurses) were enlisted for respiratory assessment during compression on the back. With the nose clipped and spontaneous breathing held, the volunteer's exhaled tidal volume upon compression was measured with a spirometer., Results: Standard external cardiac massage of the cadavers generated BPs of 55 +/- 20/13 +/- 7 mmHg; however, external compression on the back of the cadavers generated higher BP of 79 +/- 20/17 +/- 10 mmHg (p = 0.028, Wilcoxon signed-rank analysis). External compression on the back of the volunteers generated mean tidal volumes of 399 +/- 110 mL., Conclusion: Our study revealed that prone CPR provides good respiratory and circulatory support at the same time. It is easy to perform and it may be a good alternative way for bystanders to perform CPR in public surroundings. We recommend that more investigators do further studies on this topic.

Mammalian type 2 taste receptors (T2R) are a family of G protein-coupled receptors that mediate bitter signals in taste cells. In the present study, we compared the genomic organization of rodent T2R genes based on the recently completed mouse and rat genomes and examined tissue- and cell-specific expression of T2Rs. Both mouse and rat T2R families consist of 36 intact genes and at least 7 pseudogenes that are mapped to mouse chromosomes 15, 2, and 6 and to rat chromosomes 2, 3, and 4, respectively. All but two T2R genes are clustered on mouse chromosome 6 and rat chromosome 4 with virtually identical genomic organization. The orthologs of the first human T2R gene identified, mT2R119 and rT2R1, are located on mouse chromosome 15 and rat chromosome 2, whereas the novel rodent-specific T2R genes, mT2R134 and rT2R34, are located on mouse chromosome 2 and rat chromosome 3, respectively. Our results, using RT-PCR, demonstrate the presence of transcripts corresponding to the putative denatonium benzoate (DB) and phenylthiocarbamide (PTC) receptors in the antrum, fundus, and duodenum as well as in STC-1 and AR42J cells. The novel rodent-specific T2R gene (mT2R134 and rT2R34) was also expressed in these tissues and cell lines. The addition of DB, PTC, or cycloheximide to AR42J cells induced a rapid increase in the intracellular Ca(2+) concentration. The specificity of these effects is shown by the fact that these bitter stimuli did not induce any detectable Ca(2+) signaling in many other rodent or human cells that do not express receptors or G proteins implicated in bitter taste signaling. These results demonstrate that mouse and rat T2R genes are highly conserved in terms of genomic organization and tissue expression, suggesting that rodent T2Rs are evolved under similar dietary pressure and share bitter sensing functions in the lingual and gastrointestinal systems.

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.

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 CCK(A) and CCK(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-8 for displacing the label from CCK(A) and CCK(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.

I.v. injection of secretin activates neurons in brain areas controlling autonomic function and emotion. Peripheral administration of secretin inhibits gastric functions through a central mechanism that is mediated by vagal dependent pathways. We investigated whether the vagus nerve is involved in i.p. injection of secretin-induced brain neuronal activation in conscious rats as monitored by Fos immunohistochemistry. Secretin (40 or 100 microg/kg, i.p., 90 min) induced a dose-related increase in the number of Fos positive neurons in the central nucleus of the amygdala (CeA), and a plateau Fos response in the area postrema (AP), nucleus tractus solitarii (NTS), locus coeruleus (LC), Barrington's nucleus (Bar), external lateral subnucleus of parabrachial nucleus (PBel) and arcuate nucleus, and at 100 microg/kg, in the dorsal motor nucleus of the vagus (DMV) compared with i.p. injection of vehicle. Double immunohistochemistry showed that secretin (40 microg/kg, i.p.) activates tyrosine hydroxylase neurons in the NTS. Subdiaphragmatic vagotomy (7 days) abolished Fos expression-induced by i.p. secretin (40 microg/kg) in the NTS, DMV, LC, Bar, PBel and CeA, while a significant rise in the AP was maintained. In contrast, s.c. capsaicin (10 days) did not influence the Fos induction in the above nuclei. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR showed that secretin receptor mRNA is expressed in the nodose ganglia and levels were higher in the right compared with the left ganglion. These results indicate that peripheral secretin activates catecholaminergic NTS neurons as well as neurons in medullary, pontine and limbic nuclei regulating autonomic functions and emotion through vagal-dependent capsaicin-resistant pathways. Secretin injected i.p. may signal to the brain by interacting with secretin receptors on vagal afferent as well as on AP neurons outside the blood-brain barrier.

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.

Introduction and Aims: Orexins have been demonstrated to have mainly central physiological functions, including regulation of food and water intake, sleep, and arousal. However, little is known about their direct peripheral effects, if any. As a first step toward understanding the role of Orexin in non-neuronal tissues or cells, we initiated studies to examine expression of Orexin receptors (OXR) in an established pancreatic tumor cell line AR42J. Secondly, we wanted to determine whether Orexins, in various molecular forms, are active to stimulate any known pancreatic cell functions in AR42J cells., Methodology: Reverse transcription-PCR analysis was performed to identify the presence of specific Orexin receptor subtypes. Intracellular calcium mobilization and cAMP levels were measured following stimulation by Orexin A and B peptides, their respective C-terminal decapeptide fragments, and hypocretin-2-gly (glycine-extended Orexin B). Release of alpha-amylase was measured in conditioned media after acute stimulation with the set of Orexin peptides for 30 minutes. Cell proliferation was determined by H-thymidine incorporation after 24 hours following treatment with Orexins under serum-free condition., Results: RT-PCR and sequencing results showed that Orexin receptor subtype 2 (OX2R) was the main form expressed in AR42J cells. Orexins stimulated dose-dependent increases in intracellular calcium mobilization with EC50 0.05 nM for Orexin A and 0.1 nM for Orexin B but were unable to stimulate any significant cAMP accumulation or DNA synthesis even at micromolar concentrations. Both Orexin-A and -B, but not hypocretin-2-gly, also stimulated dose-dependent increases in amylase release in the AR42J cells. Orexin-A and -B carboxyl-terminal decapeptides elicited significant but much lower calcium and amylase responses., Conclusion: Our data demonstrate that OX2R mediates Ca -dependent amylase release in AR42J cells, suggesting that Orexins may have secretory functions in pancreatic tumor cells.

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of alpha subunits of G proteins implicated in intracellular taste signal transduction, namely Galpha(gust), and Galpha(t)-(2), also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca(2+) concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

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 (125)I-labeled secretin (IC(50) values 5.3 +/- 0.5 and 6.4 +/- 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 released 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.

Expression of the long form of the leptin receptor, the isoform that is considered to have full signaling capability, has been reported in the central nervous system and several peripheral cell types. However, only a few cell lines have been shown to express the long form of the receptor. AR42J, a cell line derived from azaserine-treated rat pancreas, is a common model for pancreatic acinar cell secretion. In this study, the presence of leptin-receptor variants and leptin action was evaluated in this cell line. Messenger RNAs for both the long and a short form of the leptin receptor were detected by reverse transcription-polymerase chain reaction (RT-PCR) in AR42J cells, and authenticity of the receptor was confirmed by DNA sequencing. Competitive binding studies demonstrated that binding of radiolabeled leptin was specific and did not cross-react with cholecystokinin (CCK). Biologic effects of leptin on amylase release and intracellular calcium mobilization were further assessed in the presence and the absence of CCK, a known pancreatic secretagogue. Although leptin alone (< or =200 ng/ml) did not affect basal amylase release, it inhibited amylase release stimulated by 1 nM CCK by 48%. Leptin alone had no significant effect on calcium mobilization. However, pretreatment of leptin (10 and 100 ng/ml) enhanced calcium responses stimulated by CCK. These data demonstrate that the rat pancreatic tumor cell line AR42J expresses a functional form of the leptin receptor that modulates the action of CCK in calcium mobilization and amylase release.

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.

To understand molecular basis of Gs coupling to cholecystokinin (CCK)-A and CCK-B receptor subtypes, we examined cAMP responses in three sets of human CCK receptor mutants expressed in human embryonic kidney (HEK)293 cells. Single or double substitutions of the four nonconserved amino acids in the first intracellular loop of the CCK-BR were made with their CCK-AR counterparts to determine which residues are critical in Gs coupling. Single substitution of Ser82 to Asn, produced maximal cAMP responses comparable with the chimeric CCK-BR containing the entire first intracellular loop of the CCK-AR. Two other single substitutions, Leu81 to Arg and Leu85 to Met, produced significant but smaller cAMP responses. Ser82 was further changed into Asp, Thr, or Ala to determine the specificity of this position in Gs coupling by the CCK-BR. Replacements of Ser to Asp or Thr showed significant cAMP increases but the stimulatory effects were smaller than Ser to Asn, whereas Ser to Ala did not enhance any cAMP response to either CCK or gastrin. Finally, CCK-AR reverse mutants were studied to compare them with their corresponding CCK-BR mutants that showed increased cAMP responses. Substitution of CCK-AR residue Arg68 to Leu resulted in a complete loss of cAMP response, whereas Asn69 to Ser or Met72 to Leu showed markedly diminished cAMP responses. These data identify that specific residues in the first intracellular loop of both CCK receptor subtypes are critical for Gs coupling. Substitution of a single residue Ser82 to Asn in the CCK-BR is sufficient to confer full cAMP responses to agonist stimulation.

The localization of histidine decarboxylase (HDC) activity in the enterochromaffin-like (ECL) cells of the oxyntic mucosa was studied during fasting and refeeding using monoclonal (CURE no. 44178) and polyclonal (CURE no. 94211) antibodies directed against the COOH terminus of HDC (HDC-CT). Changes in HDC immunostaining were correlated with mucosal HDC enzyme activity. Immunoneutralization of circulating gastrin and atropine treatment during refeeding were used to determine the relative importance of gastrin and cholinergic mechanisms in the regulation of HDC activity and immunostaining. Fasting caused a rapid reduction in the number of ECL cells immunostaining for HDC that was correlated with an almost complete loss of mucosal HDC enzyme activity. Refeeding restored both HDC immunostaining and enzyme activity within 2-4 h, and this response was inhibited by gastrin immunoneutralization but not by atropine treatment. Immunostaining was uniformly decreased and restored in the lower half of the oxyntic mucosa, which corresponds to the predominant area of ECL cells in the gastric gland. Histamine immunostaining and mucosal histamine content were not significantly changed during fasting and refeeding or by gastrin antibody and/or atropine treatment during refeeding. These findings indicate that HDC activity correlates with HDC-CT immunostaining and that both HDC activity and HDC-CT immunostaining are regulated by gastrin during refeeding.

Cold exposure increases TRH gene expression in hypothalamic and raphe nuclei and results in a vagal activation of gastric function. We investigated the role of medullary TRH receptors in cold (4-6 C, 90 min)-induced stimulation of gastric motor function in fasted conscious rats using intracisternal injections of TRH receptor (TRHr) antisense oligodeoxynucleotides (100 microg twice, -48 and -24 h). The gastric emptying of a methyl-cellulose solution was assessed by the phenol red method. TRH (0.1 microg) or the somatostatin subtype 5-preferring analog, BIM-23052 (1 microg), injected intracisternally increased basal gastric emptying by 34% and 47%, respectively. TRHr antisense, which had no effect on basal emptying, blocked TRH action but did not influence that of BIM-23052. Cold exposure increased gastric emptying by 64%, and the response was inhibited by vagotomy, atropine (0.1 mg/kg, i.p.), and TRHr antisense (intracisternally). Saline or mismatched oligodeoxynucleotides, injected intracisternally under similar conditions, did not alter the enhanced gastric emptying induced by cold or intracisternal injection of TRH or BIM-23052. These results indicate that TRH receptor activation in the brain stem mediates acute cold-induced vagal cholinergic stimulation of gastric transit, and that medullary TRH may play a role in the autonomic visceral responses to acute cold.

Somatostatin exerts multiple effects by activating distinct G protein-coupled receptors. Here we report the cellular sites of expression of the somatostatin subtype 2A (sst2A) receptor in the rat enteric nervous system by using a C-terminus-specific, affinity-purified antiserum and immunohistochemistry. Antibody specificity was confirmed by the cell surface staining of human embryonic kidney 293 cells expressing the sst2A receptor, the lack of staining of cells expressing the somatostatin subtype 2B receptor, and the abolition of staining by preincubating the antiserum with the C-terminus peptide used for immunization, SSt2A(361-369). The SSt2A receptor antibody recognized a broad 80 kDa band on Western blots of membranes prepared from cells transfected with sst2A receptor cDNA; following receptor membrane deglycosylation, the antibody detected an additional 40 kDa band. In the enteric nervous system, the sst2A antibody primarily stained neurons of the myenteric and submucosal plexuses, and abundant fibers distributed to the muscle, mucosa, and vasculature. Immunoreactive staining was also observed in non-neuronal cells, including presumed interstitial cells of Cajal of the intestine and enterochromaffin-like cells of the stomach. Fibers expressing sst2A receptor immunoreactivity were often in close proximity to D cells of the gastric and intestinal mucosa. Colocalization of somatostatin and sst2A receptor immunoreactivities was not observed in endocrine cells nor in enteric neurons. Double-label immunohistochemistry revealed colocalization of sst2A and vasoactive intestinal peptide immunoreactivities in enteric neurons. The multiple types of cells expressing the sst2A receptor, including enteric neurons and non-neuronal structures, in addition to the relationship between somatostatin and sst2A receptor elements, provide evidence that the sst2A receptor mediates somatostatin effects in the gastrointestinal tract via neuronal and paracrine pathways.

The intestine of the suckling rat has the unique capacity of absorbing immunoglobulins from maternal milk. We investigated intestinal Fc receptor mRNA expression and the absorption of orally administered antibodies to delineate the ontogeny and tissue specificity of this transport system. Duodenal expression of Fc receptor mRNA was at maximum levels between 1 and 19 days of age, but was not detectable during fetal life and in animals after weaning. Along the horizontal axis of the intestine, FcRn mRNA expression was maximum in the proximal duodenum and declined gradually in distal bowel. Similarly, absorption of orally administered antibody was low shortly after birth, but reached maximum levels at 14 days of age. By the time of weaning, antibody uptake had almost completely ceased. These data further delineate the temporal and spatial nature of the intestinal immunoglobulin transport system, and represent additional examples of how the intestinal Fc receptor is transcriptionally regulated.

The membrane potential (E(m)) was used as an indicator to evaluate the effect of stem cell factor (SCF) on the membrane integrity of peritoneal mast cells (PMCs). PMCs were harvested from the peritoneal lavage of Sprague-Dawley rats, purified more than 95% and cultured with or without the presence of SCF (2 x 10(-8) M). E(m) values were measured with conventional intracellular recording techniques. Results from day 1 to day 4 in culture were compared. Significant differences in average E(m) (aE(m)) (P < 0.01, analysis of variance) were seen on days 3 and 4 (means +/- SE in millivolts): -67.4 +/- 8.0 and -59.4 +/- 4.8 with SCF vs. -24.8 +/- 7.9 and -7.6 +/- 3.9 without SCF, respectively. Moreover, after culture with SCF for >1 wk, the aE(m) values of purified PMCs had a tendency to reach plateau values similar to that of unpurified PMCs on day 1 (at -20 mV). The morphological appearances of PMCs can be correlated with the results of aE(m) measurements. PMCs with a smooth spherical shape and highly refractive appearance, and better tolerance to electrode impalement, showed E(m) with greater negative values and lesser fluctuations. These results indicate that SCF can maintain the membrane properties and viability of purified PMCs in a long-term culture.

To investigate the physiological role of endogenous gastrin-releasing peptide (GRP) in regulating the release of gastrin, we evaluated the response of intragastric pH, gastrin, and GRP after omeprazole treatment in rats. A significant elevation of the plasma level of GRP (P < 0.01) and a significant reduction of the antral content of GRP (P < 0.05) were observed after the administration of 100 mg/kg omeprazole. The antral content of GRP was significantly decreased 12 h after omeprazole administration and thereafter gradually returned to control levels. Peak values for intragastric pH and plasma GRP were observed 3 h after omeprazole administration and before the peak of serum gastrin. The bombesin antagonist [D-Phe6]-bombesin-(6,13)-methyl ester significantly inhibited gastrin release after omeprazole treatment (P < 0.05). These observations indicate that omeprazole-induced inhibition of acid secretion stimulates the release of GRP and suggest that the secretion of GRP induced by omeprazole may stimulate the secretion of gastrin, at least in the early phase.

Avian gastrin is a gastric acid secretagogue, but is structurally more closely related to mammalian cholecystokinin, which is functionally distinct from gastrin. This apparently anomalous structure/activity relationship raises important issues for understanding the evolution of regulatory peptides and the mechanisms that control their expression. To clarify the possible mechanisms, we have determined the sequence of a 6.5-kb BamHI genomic DNA fragment that includes the entire avian gastrin transcriptional unit. The complete cDNA sequence, determined by anchored PCR, encoded a precursor of 105 amino acids. Conserved primary amino acid structures were limited to a decapeptide determining biological activity, and essential sites for post-translational processing. Significantly, however, the genomic regulatory regions, and introns, were unlike those of any previously reported gastrin/cholecystokinin gene. The avian gastrin gene contained no recognizable TATA-box motif, a feature unique to this gene family, but had a G+C-rich region immediately upstream of the transcription initiation site, and a Z-DNA purine-pyrimidine repeat sequence. Moreover, physiological regulation of the avian gastrin gene differed markedly from that observed in mammals, in that the important local paracrine downregulation by antral somatostatin was absent; instead, evidence for a hormonal regulation was demonstrated. The data indicate that genomic regulatory elements within regulatory peptide families such as the gastrin/cholecystokinin family, and mechanisms of physiological control, are not conserved during evolution, even though biologically important amino acid sequence information is retained.

Background/aims: Gastric enterochromaffinlike (ECL) cells play an important role in peripheral regulation of acid secretion. This study investigated the somatostatin receptor subtype on ECL cells., Methods: ECL cells were isolated from rat fundic mucosa to a purity of 90%-95% by combining enzymatic digestion, elutriation, density gradient centrifugation, and culture., Results: Polymerase chain reaction performed with templates from an ECL cell complementary DNA library and primers specific to each of the five known somatostatin receptor subtypes showed that the somatostatin receptor type 2 was significantly enriched in ECL complementary DNA. Single cell videoimaging of highly purified ECL cells in culture showed that only the somatostatin receptor type 2 selective agonist, DC 32-87, inhibited the gastrin-induced calcium signal at 10(-11) mol/L. The type 3 and type 4 selective agonists, DC 25-12 and DC 32-92, and also somatostatin 14 required 100-1000 times higher concentrations (10(-8) mol/L). The somatostatin receptor type 2 analogue also inhibited gastrin-stimulated histamine release with a 50% inhibitory concentration (IC50) value of 2 x 10(-12) mol/L, whereas somatostatin 14 and the type 3 and 4 analogues showed IC50 values of 1 to 5 x 10(-9) mol/L., Conclusions: The predominant somatostatin receptors on rat gastric ECL cells are of the somatostatin receptor 2 subtype; they inhibit histamine secretion by interfering with the gastrin-induced calcium signal.

The stimulation of thyrotropin release by cold is associated with an increase in thyrotropin-releasing hormone gene expression in the paraventricular nucleus of the hypothalamus. Cold exposure also stimulates autonomic outflow to viscera. There is evidence that caudal raphe nuclei are involved in autonomic regulation through thyrotropin-releasing hormone projections to the dorsal vagal complex and spinal cord. To determine whether cold modulates thyrotropin-releasing hormone gene expression in the caudal raphe nuclei, the effect of cold exposure on thyrotropin-releasing hormone messenger RNA levels in the rat lower brainstem was examined by quantitative Northern blot analysis and thyrotropin-releasing hormone messenger RNA was localized by in situ hybridization. The gastric responses to cold exposure were also assessed in sham or vagotomized rats with pylorus ligation. Thyrotropin-releasing hormone messenger RNA signal was detected in the RNA extracted from the medulla and hypothalamus but not from the amygdala, periaqueductal gray or cerebellum. Cold exposure (4 degrees C) for 1 or 3 h increased thyrotropin-releasing hormone messenger RNA levels in the medulla by 77 +/- 37 and 142 +/- 39% respectively. In situ hybridization histochemistry showed that the increase in silver grain density occurred exclusively in the raphe pallidus and raphe obscurus. Exposure to cold stress for 2 h stimulated gastric acid secretion and resulted in gastric lesion formation in sham but not vagotomized rats. There are established thyrotropin-releasing hormone projections from the raphe pallidus and obscurus to the dorsal vagal complex.(ABSTRACT TRUNCATED AT 250 WORDS)

This study demonstrates the effectiveness of parenteral and oral anti-somatostatin monoclonal antibody to stimulate gastrin cell activity in suckling rats. Intraperitoneal anti-somatostatin monoclonal antibody increased serum gastrin concentration (> 2-fold), and orally administered antibodies retained their neutralizing capabilities as demonstrated by a 30% induction of gastrin synthesis. Absorption of luminal monoclonal antibody was time dependent and saturable as demonstrated by measurement of serum murine immunoglobulin G and the subsequent effects on serum gastrin and antral gastrin mRNA concentrations. Enhanced gastrin synthesis after oral administration required whole antibody in that enterally delivered F(ab')2 fragments were not absorbed and did not increase serum gastrin concentrations. In addition, pretreatment with excess Fc fragments decreased monoclonal antibody absorption and eliminated the serum gastrin response to oral monoclonal antibody. These results demonstrate that orally administered murine anti-somatostatin monoclonal antibody was rapidly and efficiently absorbed via an Fc-dependent mechanism and retained immunoneutralizing activity against endogenous somatostatin, neutralizing its inhibition of gastrin cell activity. These results indicate that orally administered monoclonal antibodies could potentially be used to determine the role of other peptides and growth factors as potential physiological regulators during the suckling period of postnatal development.

Gastrin, cholecystokinin (CCK), and CCK-related peptides comprise a hormonal family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium ([Ca2+]i). Furthermore, gastrin acts as a direct growth factor through CCKB/gastrin receptors. We report here that the expression of the mRNA coding for CCKB/gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of Ca2+ mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expression of CCKB/gastrin receptor mRNA. Accordingly, gastrin (1-100 nM) did not cause any measurable increase in [Ca2+]i. In contrast, the addition of cholecystokinin residues 26-33 (CCK-8) caused a rapid and transient increase in [Ca2+]i in this cell line. CCK-8 mobilized Ca2+ in a dose-dependent manner in the nanomolar range (half-maximal stimulatory concentration = 12 nM). Furthermore, the selective CCKA antagonist CAM-1481 inhibited the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 3 nM) in GLC19 but not in H510 cells. The selective CCKB/gastrin antagonist blocked the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 cells in a dose-dependent manner in the nanomolar range, whereas over the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but not in H510 cells. Our results show, for the first time, that CCKA receptors can mediate Ca2+ mobilization and growth in SCLC cells and that SCLC cells express two distinct functional CCK receptor subtypes.

Hormonal control of immunoglobulin (Ig) absorption and of intestinal Fc receptor mRNA expression were investigated in rats to assess its potential role in the normal postsuckling inhibition of this transport system. Corticosterone and L-thyroxine therapy caused premature inhibition of the absorption of orally administered murine monoclonal antibody and of Fc receptor mRNA expression in a dose- and time-dependent manner. Low-dose corticosterone had no effect on Fc receptor mRNA synthesis after 3 d but decreased Ig transport fivefold after 7 d. High dose corticosterone resulted in a threefold reduction in Fc receptor after 3 d, and there was almost complete inhibition (> 30-fold) of transport and of Fc receptor transcript levels after 7 d. Similarly, 7 d of high-dose thyroxine decreased both serum Ig transport and Fc receptor (> 30-fold). However, adrenalectomy did not prevent the normal post-suckling declines in Ig transport or receptor synthesis. This study demonstrates that exogenous corticosteroids and thyroxine hormone inhibit Ig transport and steady-state duodenal Fc receptor mRNA levels in suckling rats. Endogenous adrenal steroids however, do not appear to be entirely responsible for the age-dependent decline in this transport system.

Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.

Regulation of somatostatin gene expression was studied in the rat gastric antrum. Antral total RNA was isolated from animals during starvation and after refeeding, or under gastric neutralization by fundectomy or by omeprazole treatment. Northern blot analysis using cRNA probe synthesized from a cloned rat somatostatin cDNA demonstrated a single hybridizing band, approximately 850 nucleotides in length, which is present in the antrum. Quantitative slot blot analyses were able to detect significant changes of somatostatin mRNA levels in total RNA as low as 5 micrograms. Somatostatin mRNA levels increased significantly after 12 hours of fasting (144% of control) and remained elevated throughout the 4-day fasting period. Upon refeeding with solid food and phenylalanine, antral somatostatin returned to the prefasted level in 2 hours. Refeeding with olive oil or saline depressed somatostatin mRNA significantly within 30 to 60 minutes but did not attain the prefasted state. Fundectomy and omeprazole resulted in maximal inhibition of antral somatostatin mRNA levels by 77% and 78%, respectively. The present in vivo results indicate that somatostatin gene expression in the stomach is regulated by luminal factors that include pH and specific nutrients. Future studies based on this phenomenon can expand knowledge of the interactions between gastric endocrine cells and the gastric environment.

Antral gastrin and somatostatin mRNA concentrations were measured in rats during gastric neutralization produced either by resection of the acid-secreting portion of the stomach (fundectomy) or by omeprazole treatment. Fundectomy caused increases in gastrin mRNA concentrations to 570% of sham control after 4 days and to 650% after 28 days. Daily administration of omeprazole resulted in significant dose- and time-dependent increases in antral gastrin mRNA concentrations. Four-day treatment with omeprazole caused threefold increased gastrin mRNA. Antral somatostatin mRNA concentrations decreased significantly after fundectomy to 66% of sham control after 4 days and to 23% after 28 days. Omeprazole produced a more profound decrease in somatostatin mRNA to 22% of the vehicle control after 4 days. Antral beta-actin mRNA concentrations did not differ significantly between control and experimental animals. Transcription of gastrin mRNA in isolated antral mucosal nuclei, measured by a nuclear run on technique, was significantly increased after omeprazole treatment in vivo. Increases in plasma and antral gastrin concentrations in response to gastric neutralization were closely associated with increases in gastrin mRNA and were accompanied by reductions in somatostatin mRNA in the antrum. However, fundectomy produced relatively greater increases in gastrin mRNA and lesser reductions in somatostatin mRNA than observed after omeprazole pretreatment.

We have examined the regulation of gene expression and post-translational processing of progastrin by starvation and feeding in rats. An oligonucleotide complementary to rat preprogastrin cDNA was used in RNA blot and hybridization analysis to measure gastrin mRNA levels. A region-specific antibody raised against the predicted amino acid sequence of the carboxyl terminal extension of progastrin was used for quantitation of progastrin peptides. The effects of starvation and of refeeding on rat antral gastrin mRNA and pro-hormone peptide levels were examined in rats starved for 48 h and after refeeding with a solid meal. Antral gastrin mRNA concentrations decreased to a plateau level (30% of the nonstarved control) after 48 h of starvation. Immunoreactive gastrin concentration decreased threefold, but progastrin processing intermediates did not decrease significantly during fasting. Following refeeding, significant increases in antral mRNA level were detected in 1 h, and peak levels were reached by 2 h (more than two times higher than starved control). There was a rapid and significant decrease in progastrin immunoreactivity within 30 min, followed by a significant increase in gastrin immunoreactivity 2 h after refeeding. These data suggest that rapid increases of blood and tissue gastrin levels in response to food may be associated with increases in both gastrin gene expression and post-translational processing of progastrin.

A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.

A simple, reliable, and computer-assisted assay has been developed to quantitate isotype-specific regulation of human immunoglobulin synthesis in vitro. The assay utilizes three separate human lymphoblast or myeloma cell lines, which secrete human immunoglobulins IgA, IgG, and IgE. Culture supernatants from 96-well tissue culture plates are then assayed for IgA, IgG, and IgE by a solid-phase enzyme-linked immunosorbent assay (ELISA) on a microtiter plate. Data collection and analysis is performed with the aid of computer programs designed for this assay. This assay has several advantages over other immunoglobulin regulation assays: no radioisotopes are used, thereby reducing cost and complexity; results are directly collected and quantified by computer analysis; the entire assay is completed in 3 days; reliability and reproducibility are increased by the use of established human cell lines; and co-culturing all three immunoglobulin-producing cell lines provides convenient internal controls for isotype specificity.