Elevated IL-33 expression is associated with pediatric eosinophilic esophagitis, and exogenous IL-33 promotes eosinophilic esophagitis development in mice.

We tested whether the T helper (Th) type 2 (Th2) cell agonist and allergenic ligand IL-33 was associated with eosinophilic esophagitis (EoE) development in a pediatric cohort and whether IL-33 protein could induce disease symptoms in mice. Biopsies from EoE patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast cells, and epithelial cell markers near blood vessels. Circulating levels of IL-33 were not increased. The time course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model and chronicity of IL-33 expression has been demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 wk and esophageal pathology was evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and upregulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, along with innate lymphoid cell group 2, Th1/17, and M2 macrophage marker genes, were increased after IL-33 application. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with pediatric EoE development and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.

Cytokine signalling via gp130 in gastric cancer.

Cytokine signalling pathways that depend on gp130 are dysregulated in several epithelial cancers including gastric cancer. It has been established that blockade of SHP2 activation of MAPK signalling results in hyperactivation of STAT3 resulting in increased cell proliferation, angiogenesis, inflammation and inhibition of both immunocyte and epithelial cell apoptosis. Additionally, key genes regulated downstream of gp130 via MAPK activation such as the stomach-specific tumor suppressor gene tff1 are suppressed, contributing to the oncogenic outcome. The main cytokine driver of gp130 signalling in the stomach is IL-11, with IL-6 having little activity in the antral stomach in which most pathology initiates. IL-11 is up-regulated in both mouse and human gastric cancer and in pre-neoplastic mucosa. A characteristic gene signature specifically associated with IL-11 drive has been observed, although the prognostic value of the signature has not yet been assessed. Infection of human or mouse stomach with Helicobacter pylori, especially that expressing the CagA cytotoxin, produces constitutive MAPK activation, but also activated STAT3 and increases IL-11 expression. The possibility of designing and utilising small molecule inhibitors of either IL-11 or STAT3 activation may be worthwhile in developing new cancer therapeutics.

[Between "pragmatic" interpretation and "disturbing" understanding: Embryonic cryopreservation for IVF patients]. / Entre lecture pragmatique et vision anxiogène : vécu de la cryoconservation embryonnaire par les patients en parcours de FIV.

OBJECTIVES: The aim of this article is to question the feeling of IVF patients towards embryonic cryopreservation, in order to understand their potential reluctance to freeze embryos and their difficulties to consider the fate of their frozen embryos once their parental project completed. METHODS: Twenty-seven semi-directive interviews with homologous IVF patients were conducted. These persons were followed in two fertility centres in Marseille. RESULTS: If all the patients interviewed have accepted embryonic cryopreservation or have accepted on principle, a majority have an ambivalent attitude towards this technique. If some share the "pragmatic" vision of professionals (embryologists, technicians and gynaecologists), they are numerous to worry about a possible deterioration of embryonic quality, or again about a disrupted order of generation. Finally, it appears that patients do not anticipate the possible fate of their frozen embryos if they are uninscribed from their parental project. CONCLUSIONS: Patients are mainly ambivalent towards embryonic cryopreservation. They prioritize different rationality depending on the situations and issues they are dealing with.

Combining an epithelial repair factor and anti-fibrotic with a corticosteroid offers optimal treatment for allergic airways disease.

BACKGROUND AND PURPOSE: We evaluated the extent to which individual versus combination treatments that specifically target airway epithelial damage [trefoil factor-2 (TFF2)], airway fibrosis [serelaxin (RLX)] or airway inflammation [dexamethasone (DEX)] reversed the pathogenesis of chronic allergic airways disease (AAD). EXPERIMENTAL APPROACH: Following induction of ovalbumin (OVA)-induced chronic AAD in 6­8 week female Balb/c mice, animals were i.p. administered naphthalene (NA) on day 64 to induce epithelial damage, then received daily intranasal administration of RLX (0.8 mg·mL(−1)), TFF2 (0.5 mg·mL(−1)), DEX (0.5 mg·mL(−1)), RLX + TFF2 or RLX + TFF2 + DEX from days 67­74. On day 75, lung function was assessed by invasive plethysmography, before lung tissue was isolated for analyses of various measures. The control group was treated with saline + corn oil (vehicle for NA). KEY RESULTS: OVA + NA-injured mice demonstrated significantly increased airway inflammation, airway remodelling (AWR) (epithelial damage/thickness; subepithelial myofibroblast differentiation, extracellular matrix accumulation and fibronectin deposition; total lung collagen concentration), and significantly reduced airway dynamic compliance (cDyn). RLX + TFF2 markedly reversed several measures of OVA + NA-induced AWR and normalized the reduction in cDyn. The combined effects of RLX + TFF2 + DEX significantly reversed peribronchial inflammation score, airway epithelial damage, subepithelial extracellular matrix accumulation/fibronectin deposition and total lung collagen concentration (by 50­90%) and also normalized the reduction of cDyn. CONCLUSIONS AND IMPLICATIONS: Combining an epithelial repair factor and anti-fibrotic provides an effective means of treating the AWR and dysfunction associated with AAD/asthma and may act as an effective adjunct therapy to anti-inflammatory corticosteroids

The role of capsulin in the morphogenesis and differentiation of fetal rat gastric mucosa.

The signals that guide the morphogenesis and differentiation of rat fetal gastric mucosa remain largely unknown. We have investigated the role of capsulin in pit/gland formation and epithelial cell differentiation in cultured stomach tissue. Embryonic day 16.5 (E 16.5) stomach tissue cultured for three days in the presence of 1 microM hydrocortisone underwent dramatic transformation, from undifferentiated, stratified cells to differentiated epithelia composed of polarised columnar cells with mucous cells and pit/glands. In the presence of capsulin antisense oligonucleotides directed against capsulin mRNA, tissues do not undergo further development. Significantly, both mucous granules and pit/gland formation were inhibited compared to capsulin sense/scrambled oligonucleotide treated controls. However, in tissues treated with specific anti-rat HGF-antiserum to neutralise secreted HGF, pit/gland formation was inhibited, but the number of mucous granules remained unchanged compared to controls treated with non-specific antiserum (mouse monoclonal cytokeratin 8 antiserum). This data suggests that capsulin may have a role in the morphogenesis of pit/glands and mucin granule formation in the developing rat gastric mucosa. We discuss the possibility that this role of capsulin may be partly mediated through the actions of HGF.

Trefoil peptides are early markers of gastrointestinal maturation in the rat.

Trefoil peptides are members of a unique family of proteins found predominately throughout the gastrointestinal tract, whose proposed functions include mucus stabilization, stimulation and/or differentiation of epithelial cells during wound repair. Recent trefoil knockout studies have reported delays in epithelial cell migration or maturation pathways together with almost a complete lack of mucus. In order to fully explore the role of trefoil peptides in gastrointestinal maturation, these studies were undertaken to accurately characterize the expression of trefoil peptides in the developing rat gut. The results of RPA suggest that trefoil mRNA's are expressed as early as 15 days post coitus (dpc) in the intestine and stomach. Proteins are detected at 17 dpc by radioimmunoassay and immunohistochemical studies, which localize trefoil peptide expression to the lumenal surface of epithelial cells. At 17 dpc the gut is lined by pseudo-stratified, undifferentiated epithelial cells. Polarized, columnar cells are not detected until at least 18 dpc, with sparse mucus staining and parietal cell markers not being detected until 18 and 19 dpc respectively. This data demonstrates that trefoil peptides are early markers of epithelial cell maturation in the developing rat gut. The time course of their expression, well before the mucus cell type is specified, suggests a potential role in epithelial cell differentiation.

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

Generation of Met-enkephalin Arg6Phe7 immunoreactivity by proteolytic cleavage of mammalian plasma precursors by pepsin.

A region-specific antiserum raised against the C-terminal heptapeptide of proenkephalin A (Met-enk Arg6Phe7) was used in RIA studies to show that rat, human, and ovine plasma contain substrates (mol wt, 68K) that yield nanomolar amounts of Met-enk Arg6Phe7 (ME-RF) after treatment with pepsin under acid conditions. This ovine plasma-derived immunoreactivity diluted in parallel to the ME-RF standard in RIA and chromatographed as two low mol wt species (approximately 1K) which were less hydrophobic than the standard on size exclusion and reverse phase chromatography. The pepsin-generated material displaced [3H]naloxone from rat brain binding sites; its potency was about 1000-fold that of ME-RF, assuming near 100% cross-reactivity with the antiserum. Taken together these observations suggest that the pepsin-generated material is of similar mol wt and amino acid sequence to ME-RF, but differs with respect to opiate-binding efficacy, and that the plasma precursor is distinct from proenkephalin in both size and processing sites.

Expression of gastrin-releasing peptide (GRP) and GRP receptors in the pregnant human uterus at term.

Synthesis of both gastrin-releasing peptide (GRP) and messenger ribonucleic acid (mRNA) has been demonstrated in pregnant sheep, but studies in women have not been reported. Therefore, we examined the uterus and placenta of pregnant women at term for synthesis of GRP and expression of GRP receptor genes BRS-3 and GRP-R. A transcript of 0.95 kilobases, corresponding to GRP mRNA, was detected in endometrium and myometrium, but not in amnion, chorion, placenta, or nonpregnant endometrium. GRP immunoreactivity (GRP-ir) was detected in half (three of six) of the endometrial (1.23 +/- 0.04 pmol/g) and myometrial (0.73 +/- 0.04 pmol/g) samples and in some, but not all, samples of amnion (one of four subjects; 0.6 pmol/g), chorion (four of five subjects; 0.8 +/- 0.2 pmol/g), placenta (two of six subjects; 0.5 +/- 0.2 pmol/g), and amniotic fluid (four of six subjects; 59 +/- 19 fmol/mL). GRP-ir was present in the maternal circulation (44 +/- 12 fmol/mL) and was higher in plasma obtained from the umbilical artery (152 +/- 14 fmol/mL) and vein (143 +/- 24 fmol/mL). The major peak of GRP-ir in pregnant endometrial tissue was larger than GRP-(1-27), as determined by gel filtration chromatography. Minor peaks were also observed: two larger than the main form and one corresponding to GRP-(18-27). mRNA for GRP receptors GRP-R and BRS-3 was detected by semiquantitative reverse transcription-PCR. For both receptors, mRNA was higher in the pregnant endometrium than in the nonpregnant endometrium but was detected in all of the uteroplacental tissues examined. GRP-R mRNA predominated in the pregnant endometrium, whereas BRS-3 mRNA predominated in the membranes and placenta. In these tissues, PCR for BRS-3 mRNA gave rise to an additional product (approximately 50 bp larger). These studies demonstrated that a peptide larger than, but related to, GRP is synthesized in the pregnant human uterus and is secreted into the maternal and fetal circulations. The detection of mRNA for GRP-R, BRS-3, and possibly a transcript variant of BRS-3 as well as the detection of a peptide larger than, but related to, GRP suggest a novel regulatory unit in the human reproductive tract.

Identification of the rat homologue of the mouse capsulin gene by cDNA representational difference analysis.

Representational difference analysis of cDNA (cDNA RDA) is a PCR-based differential cloning method. We have found that this PCR-based subtraction technique does not have any bias towards smaller DpnII-generated fragments. We have successfully used this method to identify the rat homologue of the mouse capsulin gene.

The trefoil peptides TFF2 and TFF3 are expressed in rat lymphoid tissues and participate in the immune response.

Members of the trefoil factor (TFF) family are mucin-associated polypeptides that are expressed along the entire length of the gastrointestinal tract. TFFs have been proposed to play a role in mucosal defence through both protective and reparative mechanisms. The potential relationship between TFFs and mucins in non-gut glycoprotein-secreting epithelia has not been fully explored. In the present study we identified TFF2 and TFF3 mRNA and peptide in rat lymphoid tissues, demonstrated that TFF peptide expression in rat spleen increased 1.5- to 3-fold following experimental induction of the immune response, and showed that hTFF2 and hTFF3 (1-5 mg/ml) stimulated migration of human monocytes. Our data suggest that TFFs may in part be involved in the repair of injury through the modulation of the inflammatory response.

Molecular cloning, genomic organization and selective expression of bombesin receptor subtype 3 in the sheep hypothalamus and pituitary.

The bombesin receptor subtype 3 (BRS-3) is considered an orphan receptor as it has a low affinity for bombesin-like peptides and no identified natural ligand. We have reported a novel form of gastrin-releasing peptide (GRP) present in high abundance in the pregnant uterus of women and sheep. As BRS-3 was originally cloned from guinea pig uterus, we postulated that the uterine GRP-like peptide may be its natural ligand. We have therefore cloned the gene for the sheep homologue of BRS-3 and determined its distribution. The sheep BRS-3 gene spans 4 kbp and comprises three exons with intron-exon borders at positions similar to those observed for the human and mouse BRS-3 genes. The predicted amino acid sequence of ovine BRS-3 has approximately 85% identity with the human, mouse and guinea pig receptors. Highly conserved amino acids important in mediating receptor G-protein coupling to second messengers and important in ligand binding were found to be conserved in ovine BRS-3. One potentially important deviation was noted: ovine BRS-3 possesses an arginine residue at position 294 instead of a histidine residue as found in all other BRS-3. His(294) was previously identified as important in ligand-receptor interactions while Arg(294) was implicated in high ligand affinity. Thus ovine BRS-3 may have binding characteristics different from those of the human, mouse and guinea pig BRS-3 receptors. In the ewe, BRS-3 mRNA expression was detected in pituitary and hypothalamus but not in tissues of the pregnant uterus (endometrium, myometrium, chorioallantois or amnion). Nor was BRS-3 expression detected in the non-pregnant uterus or in testis. This pattern of BRS-3 expression is similar to that observed in the mouse but different from that observed in the human, rat and guinea pig. We conclude that there is no local interaction between uterine GRP-like peptide and BRS-3. However, the high expression of BRS-3 in the pituitary coupled with elevated circulating levels of this GRP-like peptide during pregnancy suggests an alternate pathway. Cloning of the ovine BRS-3 gene will permit a detailed functional analysis of this receptor in the sheep and its role in the mediation of action of uterine GRP.

Isolation and characterisation of the ovine gastrin-releasing peptide gene; abundant expression in the pregnant uterus and selective expression in fetal tissues.

High concentrations of a peptide related to gastrin-releasing peptide (GRP) are produced in the utero-placental unit of the human and sheep and secreted into the general circulation. This suggests an endocrine role in addition to its role as a neurotransmitter/neuromodulator. The GRP is larger than the previously described form GRP(1-27) but it is not known whether the larger form is the product of a related GRP-like gene or differences in post-translational processing. We have therefore cloned the gene for the sheep homologue of the GRP gene and determined its distribution. Only a single GRP gene was found in the sheep. This had a similar organisation to the human GRP gene with three exons and two introns. The larger form of GRP in the pregnant endometrium therefore appears to be the result of an alteration in processing of the GRP prohormone. The expression of GRP mRNA in the pregnant uterus was extraordinarily high comprising one-third of all mRNA synthesised by the pregnant endometrium. As the endometrial GRP mRNA arises solely from the glandular epithelium, the localised synthesis of GRP mRNA would be far higher. GRP mRNA was expressed in a wide variety of fetal tissues (fundus, colon, jejunum, ileum, duodenum, kidney, adrenal, lung, heart and pancreas) with a corresponding presence of GRP immunoreactivity. The expression of GRP in the fetal lung was biphasic with peaks at mid-term and near parturition but none in the adult supporting the concept of a specific developmental role of GRP in the lung.

Temporal expression and cellular localization of a gastrin-releasing peptide-related gene in ovine uterus during the oestrous cycle and pregnancy.

Synthesis of both mRNA and peptide for gastrin-releasing peptide (GRP) has been demonstrated in the pregnant endometrium of sheep and women. However, it is not known whether GRP is synthesized in the sheep uterus during the oestrous cycle. Furthermore the cellular site of GRP mRNA synthesis in the uterus has not been determined. Therefore we examined the synthesis of GRP and determined the cellular location of GRP peptide and mRNA in sheep uterus taken at different times during the oestrous cycle (duration 17 days) and pregnancy (duration 145 days). Northern blot analysis of RNA isolated from ovine endometrium revealed low or no GRP mRNA at days 4, 10, 12 and 14 of the oestrous cycle and a 24-fold rise in GRP mRNA (normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA) between days 14 and 16. A similar pattern was observed during early pregnancy, with a 12-fold rise in GRP mRNA:GAPDH mRNA between days 17 and 20 of pregnancy. Levels of GRP peptide were determined by RIA and found to be low in endometrium isolated at days 4, 10, 12 and 14 of the oestrous cycle (1.0-1.6 pmol/g) and 4 to 5-fold higher at day 16. In situ hybridization localized GRP synthesis to the epithelial cells of the uterine glands at day 16 of the oestrous cycle and at days 17, 20, 40 and 50 of pregnancy. At day 140 of pregnancy diffuse hybridization to cells of the myometrium was also observed. Immunohistochemistry localized GRP peptide to the apical cytoplasm of uterine glandular epithelial cells at day 16 of the oestrous cycle. For samples obtained at day 20 of pregnancy, the area surrounding the glands also showed moderately strong staining. Further staining in the glandular lumen and the stromal tissue surrounding the glands was apparent at day 140 of pregnancy. No GRP immunoreactivity could be detected in the peripheral plasma during the oestrous cycle or the first 20 days of pregnancy. Sizing chromatography of GRP immunoreactivity extracted from endometrial tissue taken at day 10 of the oestrous cycle revealed two peaks that co-eluted with GRP(1-27) and GRP(18-27). However, during luteolysis and oestrus the major peak of GRP immunoreactivity extracted from endometrial tissue was larger than GRP(1-27) and similar to that seen previously in the gravid ovine endometrium. These studies demonstrate that a peptide similar to, but larger than, GRP is a major product of the glandular epithelium of the ovine uterus during the luteal regression phase of the oestrous cycle and post-blastocyst implantation in pregnancy and provide further evidence that GRP-related peptides have important regulatory roles in uterine function.

Tissue-specific regulation of gastrin-releasing peptide synthesis, storage and secretion by oestrogen and progesterone.

In the ovine endometrium, dramatic increases in gastrin-releasing peptide (GRP) mRNA and immunoreactivity are observed during the luteal regression phase of the oestrous cycle (24-fold) and during pregnancy (at least 150-fold). This study sought to determine whether oestrogen and/or progesterone were responsible for the temporal regulation of GRP observed in the uterus. Ovariectomized sheep were divided into four groups (n=4), as follows: 1, untreated; 2, given subcutaneous and intravaginal progesterone implants; 3, given subcutaneous oestrogen implants; and 4, treated with both oestrogen and progesterone. After 10 days, the animals were sacrificed and plasma, pituitary and endometrium were obtained. A fifth group of sheep with intact ovaries was included. Analysis of endometrial GRP-immunoreactivity (GRP-ir) revealed a twofold drop for groups treated with oestrogen, progesterone or both hormones. A dramatic reduction in endometrial GRP mRNA was o! bserved in the group treated with both hormones. GRP-ir was measured in whole pituitaries and found to vary greatly (1.7-53.7 pmol/g tissue) within all groups of ovariectomized animals. There were no significant differences between any of the five groups. A significant reduction in circulating GRP-ir was observed after 10 days of treatment with either oestrogen or progesterone. These studies demonstrate that, in sheep, the synthesis, storage and secretion of GRP are differentially affected by oestrogen and progesterone. Regulation appears to be tissue specific since GRP content in the pituitary is unchanged by oestrogen or progesterone whereas GRP expression in the endometrium is inhibited. Changes in GRP mRNA expression did not correlate with changes in endometrial expression of mRNA for oestrogen receptor alpha, oestrogen receptor beta and the progesterone receptor. This study is the first reported demonstration that expression of the GRP gene can be influenced by the presence of ovarian steroids, with the conclusion that oestrogen and/or progesterone act as negative regulators of endometrial GRP expression.

Distribution and molecular forms of immunoreactive bombesin in the ovine median eminence.

The distribution and molecular forms of bombesin-like immunoreactivity (BLI) were determined in the ovine median eminence using a new, C-terminally directed antiserum. BLI was confined to neurons of the external zone of the median eminence, near small blood vessels, many of which also were immunoreactive for CRF. Each median eminence contained about 10 pmol of BLI (533 pmol/g tissue). Gel filtration and reverse-phase high pressure liquid chromatography (HPLC) demonstrated the existence of two molecular forms of BLI, which co-eluted with porcine gastrin releasing peptide (GRP)1-27 and GRP18-27 in a molar ratio of 1:2. The presence of BLI in the ovine median eminence and its co-localization in some neurons with CRF, suggest a possible role for peptides of the bombesin family in the regulation of pituitary function.

Distribution, Isolation and Sequence Analysis of the C-Terminal Heptapeptide of Pro-Enkephalin A (YGGFMRF) from the Ovine Median Eminence.

Abstract Using a polyclonal antiserum raised against the C-terminal heptapeptide of pro-enkephalin A, we have isolated the opioid heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe (MERF) from ovine median eminence and mapped its distribution in that structure. MERF-immunoreactivity was confined to the pars externa (neurosecretory zone) where it colocalized with corticotrophin-releasing factor in the majority of terminals. No larger, N-terminally extended forms of MERF were detected in median eminence extracts suggesting that pro-enkephalin is fully processed to its constituent enkephalin congeners, and that the bioactive products, including MERF, act at the level of the hypothalamus in regulating anterior pituitary function.

Distribution and chromatographic characterization of peptide tyrosine leucine amide (PYLa) immunoreactivity in mammalian tissues.

Using a polyclonal antiserum raised against the mid-region of the abundant amphibian skin peptide PYLa (peptide tyrosine leucine amide) we have shown that immunoreactive PYLa is present in a wide variety of rat tissues, being particularly abundant in the liver, spleen and gut. In each tissue the predominant molecular form corresponds to a moderately hydrophobic, basic peptide of about 10 amino acids. Using the same antiserum in immunohistochemistry we have demonstrated that immunoreactive PYLa was present in endocrine cells, but not neurons, in the ileal mucosa of rat and man. These findings are consistent with an hormonal or local tissue regulatory role for immunoreactive PYLa in the intestine.

Bombesin regulation of adrenocorticotropin release from ovine anterior pituitary cells.

Mammalian members of the bombesin-like peptide family (gastrin releasing peptides; GRP) have been localized in the ovine median eminence and in hypophysial-portal blood, suggesting a role in the regulation of anterior pituitary function. In this study we have shown that although bombesin cannot stimulate ACTH secretion alone, it potentiates release by ovine CRF, an effect blocked by the GRP receptor antagonist D-Tyr6bombesin (6-13) propylamide. Bombesin did not potentiate AVP-stimulated ACTH release; instead release was attenuated when bombesin was given at a 10-fold or greater molar excess over AVP, with no interaction seen at lower concentrations. We conclude that ovine corticotrophs express bombesin receptors, and that GRP may act in concert with other hypothalamic releasing factors to regulate ACTH secretion.

Characterization of bombesin receptors on canine antral gastrin cells.

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)