Biomedical subjects

J H Walsh

Publications and source records attributed to J H Walsh.

At least 73 records · Page 4Linked to original sources

PubMed1997

Somatostatin 2A receptor is expressed by enteric neurons, and by interstitial cells of Cajal and enterochromaffin-like cells of the gastrointestinal tract.

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.

Amino Acid Sequence↗

PubMed1997

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways.

ADP Ribose Transferases↗

PubMed1997

First intracellular loop of the human cholecystokinin-A receptor is essential for cyclic AMP signaling in transfected HEK-293 cells.

Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous members of the seven transmembrane domain G-protein-coupled receptor superfamily. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells. Chimeric receptors that maintained high affinity binding to CCK exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK-BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.

Adenylyl Cyclases↗

PubMed1997

Peptide YY inhibition of rat gastric enterochromaffin-like cell function.

BACKGROUND & AIMS: The cellular target for peptide YY (PYY) inhibition of gastric acid secretion is unknown. The aim of this study was to determine whether PYY inhibits histamine release from isolated enterochromaffin-like (ECL) cells by stimulation of a selective Y receptor. METHODS: Isolated rat gastric ECL cells were analyzed in short-term culture for histamine release and for changes in intracellular calcium concentration using video imaging. RESULTS: Gastrin-stimulated histamine release was inhibited with a 50% inhibiting concentration of 2.10(-9) mol/L. Inhibition of histamine release and of calcium entry by PYY and [Pro34]PYY and no effect of PYY(3-36) characterizes an inhibitory Y1 receptor subtype. Reverse-transcription polymerase chain reaction of ECL cell RNA showed that the receptor was the nontruncated Y1 isoform. The inhibitory action of PYY and related peptides on gastrin-stimulated histamine release and calcium signaling was eliminated by pretreatment with 200 ng/mL pertussis toxin. Additive but not synergistic inhibitory effects of PYY and somatostatin on gastrin-stimulated histamine release were observed. CONCLUSIONS: Activation of a Y1 inhibitory receptor subtype present on the gastric ECL cell that inhibits gastrin-induced ECL cell histamine release and Ca2+ entry by activation of a Gi or G(o) class of protein may account for inhibition of gastric acid secretion by PYY released from the small intestine.

Animals↗

PubMed1997

Gastrin-releasing peptide-induced expression of prostaglandin synthase-2 in Swiss 3T3 cells.

Prostaglandins, produced in response to mitogens and cytokines, are potent modulators of gastrointestinal physiology and pathophysiology. We investigated modulation of Prostaglandin synthase 2 (PGS-2) expression by the gastrin-releasing peptide (GRP) receptor in Swiss 3T3 cells. PGS-2 mRNA expression in Swiss 3T3 cells was determined by Northern blot analysis. PGS-2 protein expression in Swiss 3T3 cells was measured by Western blot analysis. GRP caused a transient induction of PGS-2 mRNA in Swiss 3T3 cells that resulted in GRP-dependent expression of PGS-2 protein. Transcriptional activation of PGS-2 by GRP was independent of de novo protein synthesis and was not affected by pertussis toxin. Comparison of signaling pathways used by PMA or EGF to those used by GRP showed that PGS-2 induction by GRP increased under conditions that inhibit PKC activity. Dexamethasone, which blocks PMA and EGF induction of PGS-2, also inhibited GRP-induced accumulation of PGS-2 mRNA. These results show that PGS-2 expression in Swiss 3T3 cells is not only controlled by PKC and receptor tyrosine kinase pathways but also by G-protein coupled receptor signaling pathways.

3T3 Cells↗

PubMed1997

Effect of enprostil on omeprazole-induced hypergastrinemia and inhibition of gastric acid secretion in peptic ulcer patients.

This study was performed to examine the effects of additional enprostil administration on hypergastrinemia and gastric acid suppression induced by omeprazole. Serum gastrin concentrations were measured in 10 peptic ulcer patients (six Helicobacter pylori-positive and four Helicobacter pylori-negative patients) before treatment, after two weeks of omeprazole (20 mg/day), and after two weeks of omeprazole and enprostil (50 micrograms/day). The additional acid inhibitory effect of enprostil was evaluated by 24-hr intragastric pH measurements in five healthy Helicobacter pylori-negative volunteers. After omeprazole treatment, the serum gastrin level of Helicobacter pylori-positive patients (3.5-fold of control) was markedly higher than that of Helicobacter pylori-negative patients (1.7-fold of control). Additional treatment with enprostil suppressed serum gastrin levels to 0.4-fold and 0.7-fold of omeprazole treatment levels in Helicobacter pylori-positive and Helicobacter pylori-negative patients, respectively. In healthy volunteers, median pH recorded during the nonmeal daytime interval increased significantly with additional enprostil. Thus, enprostil reduces undesirable omeprazole-induced hypergastrinemia, especially in Helicobacter pylori-positive patients, and effectively suppresses acid secretion.

Adult↗

PubMed1997

Ontogenetic development and distribution of antibody transport and Fc receptor mRNA expression in rat intestine.

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.

Animals↗

PubMed1997

Immunohistochemical localization of gastrin/CCK-B receptors in the dog and guinea-pig stomach.

Gastrin/CCK-B receptors are involved in the regulation of several types of cells of the gastric mucosa, including the parietal cells, the ECL cells and the D cells. In this study, we aimed at localizing such receptors in the gastric mucosa. For this purpose, we prepared monospecific antibodies against two sequences of the canine gastrin/CCK-B receptor. Sections of formalin-fixed, paraffin-embedded corpus and antrum from dog and guinea-pig were immunostained with these antibodies. In parallel, sections were stained with antibodies against somatostatin. Staining with gastrin/CCK-B receptor antibodies was observed in a few, small epithelial cells in the bottom part of the corpus mucosa. Immunoreactive cells of the antral mucosa were structurally similar, but more frequent. The same cells also stained with somatostatin antibodies. In addition one of the gastrin/CCK-B antibodies reacted with canine submucosal smooth muscle cells. No staining was observed in sections exposed to antibodies that were pre-absorbed with the corresponding antigen. We conclude that gastrin/CCK-B receptors are present in D cells of the gastric mucosa and in submucosal smooth muscle cells.

Animals↗

PubMed1997

Candidate canine enterogastrones: acid inhibition before and after vagotomy.

The relative contributions of several gut-derived peptides as enterogastrones known to be released in response to a fatty meal and to inhibit acid secretion have not previously been compared directly. We determined the acid-inhibitory activities of increasing intravenous doses of several peptides before and after highly selective vagotomy (HSV) during intragastric titration of a peptone meal in dogs. Before HSV, threshold inhibitory doses of peptide YY (PYY), cholecystokinin (CCK), and secretin were 5, 7, and 10 pmol.kg-1.h-1, respectively, whereas neurotensin, glucagon-like peptide-1 (GLP-1), and oxyntomodulin failed to inhibit acid secretion at doses up to 1,000 pmol.kg-1.h-1. The calculated dose producing 50% acid inhibition (ID50) of secretin (62 pmol.kg-1.h-1) was one-half that of PYY (128 pmol.kg-1.h-1). Maximal (90%) acid inhibition was produced by 100 pmol.kg-1.h-1 secretin and 500 pmol.kg-1.h-1 PYY. The highest dose of CCK that did not cause vomiting (100 pmol.kg-1.h-1) inhibited peptone-stimulated acid output by only 60%. After HSV, 500 pmol.kg-1.h-1. PYY and 200 pmol.kg-1.h-1 CCK failed to inhibit acid output by more than 50%. Threshold doses for inhibition by PYY and CCK were 200 and 100 pmol.kg-1.h-1, respectively. Secretin remained a potent inhibitor after HSV, with an ID50 of 80 pmol.kg-1.h-1 and a threshold dose of 10 pmol.kg-1.h-1. HSV also failed to affect inhibition caused by somatostatin. This study has shown that PYY and secretin are somewhat more potent and efficacious inhibitors of acid secretion than CCK but that all three peptides are far more active than GLP-1, neurotensin, and oxyntomodulin. PYY and CCK inhibit acid secretion in large part through vagal innervation of the gastric fundus, but the inhibitory effects of secretin are independent of fundic vagal innervation.

Animal Feed↗

PubMed1997

Somatostatin inhibits gastrin release and acid secretion by activating sst2 in dogs.

Somatostatin is a potent inhibitor of gastrin-stimulated acid secretion by activation of somatostatin receptor type 2 (sst2) in vivo, probably in part by blocking gastrin-stimulated histamine release from enterochromaffin-like cells expressing sst2. We propose that activation of sst2 may also regulate meal-stimulated acid secretion by blocking gastrin release from antral G cells. Using peptide analogs relatively selective for sst2 (NC-8-12), sst3 (BIM-23058), and sst5 (BIM-23052), we tested this hypothesis in two ways: first, in vivo by measuring plasma gastrin release during meal-stimulated acid secretion in dogs, and second, in vitro by measuring bombesin-stimulated gastrin release from an enriched culture of canine antral G cells. In vivo, a low dose (0.05 nmol.kg-1.h-1) of NC-8-12 inhibited acid secretion 56 +/- 16% without blocking gastrin release. A higher dose (1 nmol.kg-1.h-1) of NC-8-12 abolished acid secretion and inhibited gastrin release by 61 +/- 4%, whereas the highest dose (5 nmol.kg-1.h-1) inhibited gastrin release by 84 +/- 3%. Only the highest doses (5 nmol.kg-1.h-1) of BIM-23058 and BIM-23052 significantly inhibited gastrin release and acid secretion. In vitro, NC-8-12 (10(-9) M) reduced bombesin-stimulated gastrin release from antral G cells by 49 +/- 5%, whereas BIM-23058 and BIM-23052 were at least 100-fold less effective. These results indicate that somatostatin activation of sst2, but not sst3 or sst5, is the major pathway for somatostatin-induced inhibition of meal-stimulated gastrin release and acid secretion.

Animals↗

PubMed1997

Inhibition of sham feeding-stimulated acid secretion in dogs by immunoneutralization of gastrin.

A monoclonal antibody to gastrin was used to study the role of circulating gastrin in mediating acid secretion stimulated by sham feeding in dogs. On separate days, four conscious, fasted, adult mongrel dogs with esophageal and gastric fistulae were pretreated intravenously with either 7 mg of gastrin monoclonal antibody (MAb 28.2), 7 mg of keyhole limpet hemocyanin monoclonal antibody as control, or 12.5 micrograms/kg atropine sulfate. Thirty minutes later, acid secretion was stimulated first by sham feeding for 5 min, then, 60 min later, by an intravenous infusion of a maximum stimulatory dose of histamine (40 micrograms/kg) for 60 min, and after returning to basal, by intravenous infusion of a submaximal stimulatory dose of gastrin (200 pmol.kg-1.h-1) for 60 min. Acid output from secretions collected every 15 min by gravity drainage was determined by titration to pH 7.0 with 0.2 N NaOH. Sham feeding-stimulated acid output (17.7 +/- 5.5 mmol/h) was significantly inhibited by administration of either MAb 28.2 (0 mmol/h) or atropine (1.7 +/- 1.1 mmol/h). Histamine-stimulated acid output (19.6 +/- 3.4 mmol/h) was not reduced by either pretreatment. Gastrin-stimulated acid output (3.9 +/- 0.6 mmol/h) was significantly reduced only by pretreatment with MAb 28.2 (0.1 +/- 0.1 mmol/h) and not by atropine (2.2 +/- 1.4 mmol/h). A background intravenous infusion of pentagastrin (0.5 microgram.kg-1.h-1) restored sham feeding-stimulated acid output blocked by administration of MAb 28.2, although the intrinsic acid response to sham feeding could not be seen with the background pentagastrin infusion. Furthermore, the plasma gastrin response to sham feeding was not blocked by atropine pretreatment. Because immunoneutralization of both gastrin and cholinergic blockade significantly inhibited acid output during sham feeding, circulating gastrin and cholinergic pathways are involved in mediating the cephalic phase of gastric acid secretion in dogs.

Animals↗

PubMed1997

Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture.

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for canine fundic D cells. Bombesin (EC50 6 X 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 X 10(-10) M, for both) were almost equally potent stimuli of somatostatin release, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2+)-phospholipid-dependent signal-response transduction, as is indirectly suggested by potentiating interactions with DBcAMP or epinephrine.

Animals↗

PubMed1996

Distribution of neurokinin-2 receptors in the guinea-pig gastrointestinal tract.

The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine.

Animals↗

PubMed1996

Gastrin partially mediates insulin-induced acid secretion in dogs.

A monoclonal antibody to gastrin was used to study the role of circulating gastrin in mediating insulin-stimulated acid output. On separate days, seven adult dogs with chronic gastric fistulas were pretreated i.v. with either 1) 7 mg of a gastrin monoclonal antibody (mAb 28.2); 2) 12.5 micrograms/kg atropine; 3) mAb 28.2 and atropine together; or 4) vehicle (0.1% canine serum albumin in 0.15 M NaCl). Thirty minutes later, acid secretion was stimulated by insulin (0.5 U/kg, i.v.), followed in 2 h by a 1-h infusion of histamine (40 micrograms/kg/h, i.v.). Acid output (mmol/15 min) in gastric effluent collected through the gastric fistula was determined by titration with 0.2 N NaOH to pH 7.0. Plasma gastrin was measured by radioimmunoassay. Plasma glucose was measured by a glucose oxidase method on an auto analyzer. Insulin induced a profound hypoglycemia (55 +/- 8 mg/dl) that coincided with a marked increase in acid output to 7.1 +/- 0.6 mmol/30 min by 45 min after injection. MAb 28.2 pretreatment and atropine pretreatment reduced insulin-stimulated acid outputs to 2.7 +/- 0.7 mmol/30 min and to 0.6 +/- 0.2 mmol/ 30 min, respectively. Acid output after combined pretreatment (0.5 +/- 0.2 mmol/30 min) was not significantly different than after atropine alone. Histamine-stimulated acid output (15.8 +/- 2.5 mmol/30 min) was not significantly reduced by any pretreatment. Insulin injection increased circulating gastrin concentrations to 32 +/- 7 fmol/ml, which was not significantly affected by atropine (39 +/- 9 fmol/ml). This study demonstrates that, in dogs, a significant part of insulin-stimulated acid secretion is mediated by circulating gastrin.

Animals↗

PubMed1996

Effects of ranitidine bismuth citrate on gastric acid secretion and gastrin release in subjects with and without Helicobacter pylori infection.

BACKGROUND: Ranitidine bismuth citrate is a novel antiulcerant that provides the antisecretory activity of ranitidine and the gastric mucosal protection and antibacterial properties of bismuth. METHODS: This randomized, double-blind, placebo-controlled study evaluated the effects of single doses of ranitidine bismuth citrate 200 mg, 400 mg and 800 mg and ranitidine hydrochloride 150 mg on gastrin release and suppression of gastric acid secretion, and compared acid secretory profiles and gastrin release between Helicobacter pylori-negative and -positive patients. Plasma gastrin concentrations were determined by radioimmunoassay under basal conditions and in response to peptone meal stimulation. Acid secretion was measured under basal conditions and in response to peptone meal stimulation. Presence of H. pylori was determined by both 14C-urea breath test and ELISA serology. RESULTS: Inhibition of gastric acid output by ranitidine bismuth citrate was both time- and dose-dependent over the 9-h post-dose study period. Doses of ranitidine bismuth citrate 400 mg and ranitidine hydrochloride 150 mg, which are equimolar, produced similar suppression of acid output regardless of H. pylori status. Ranitidine bismuth citrate had no effect on plasma gastrin concentrations regardless of H. pylori status. All doses of ranitidine bismuth citrate were well tolerated. CONCLUSIONS: Ranitidine bismuth citrate caused time- and dose-dependent reductions in meal-stimulated and between-meal gastric acid output regardless of H. pylori status. The magnitude of decreased acid secretion was similar with ranitidine bismuth citrate 400 mg and ranitidine hydrochloride 150 mg. Ranitidine bismuth citrate had no effect on plasma gastrin concentrations.

Administration, Oral↗

PubMed1996

Selective ligand-induced intracellular calcium changes in a population of rat isolated gastric endocrine cells.

BACKGROUND & AIMS: Peripheral regulation of acid secretion depends mainly on stimulation or inhibition of the three major gastric endocrine cells (enterochromaffin-like, gastrin, and somatostatin). The aim of this paper was to define physiological responses of enterochromaffin-like, gastrin, and somatostatin cells in a mixed endocrine cell population by measuring ligand-selective changes of intracellular calcium ([Ca2+]i) in individual cells. METHODS: Endocrine cells were enriched from a rat gastric cell suspension by elutriation, a density-gradient fractionation, and a 48-hour short-term culture. [Ca2+]i responses of individual cells to various ligands such as gastrin/carboxy-terminal cholecystokinin octapeptide and selective cholecystokinin antagonists, carbachol, and gastrin-releasing peptide were monitored using video imaging in a perfusion chamber. Characteristic [Ca2+]i changes distinguished the three cell types, confirmed by immunostaining. RESULTS: All enterochromaffin-like cells respond to cholecystokinin-B receptor stimulation, but only a few respond to carbachol. Gastrin cells respond to both gastrin-releasing peptide and carbachol but not to cholecystokinin-receptor agonists. Somatostatin cells have both stimulatory cholecystokinin-A and cholecystokinin-B receptors and inhibitory muscarinic receptors. All cells have inhibitory somatostatin receptors. CONCLUSIONS: Calcium-signaling responses of gastric endocrine cells are distinctive. This allows individual cell types in a mixed population to be characterized and permits an analysis of the hormones and transmitters that act directly on a specific cell type.

Animals↗

PubMed1996

Monoclonal antibody to VIP: production, characterization, immunoneutralizing activity, and usefulness in cytochemical staining.

A very stable cell line has been generated that produces monoclonal antibody to VIP designated as CURE.V55. This hybridoma was produced by fusion of spleen cells from an immunized Robertsonian mouse containing the translocated 8.12 chromosome with FOX-NY myeloma cells that are APRT deficient. VIP monoclonal antibody producing cell line #55 was selected by limiting dilutions using thymocytes as feeder layers. Ascites fluid containing high concentration of VIP monoclonal antibody was produced from pristane-primed BALB/c mice. Ascites fluid contained approximately 20 mg/ml IgG and bound 50% of 2 fmol 125I-VIP at a final dilution of 1:50,000. Binding of this IgG1 antibody was inhibited by 50% at 5 nM concentration of either VIP 1-28 or VIP 7-28. Protein-A purified IgG of this antibody, used in a concentration of 30 mg/kg per rat (IV), completely reversed the inhibitory effect of gastric corpus contractions induced by intravenous injection of VIP (10 nmol/kg) in sodium pentothal-anesthetized rats. A control anti-keyhole limpet hemocyanin monoclonal antibody did not alter the stimulatory effect of VIP on gastric corpus contractions. Immunohistochemistry showed that this VIP monoclonal antibody stains neurons, and nerve fibers in human and rat gallbladder and the sphincter of Oddi as previously described with our polyclonal antiserum.

Animals↗

PubMed1996

Secretion of type II phospholipase A2 and cryptdin by rat small intestinal Paneth cells.

We examined the secretion of antimicrobial proteins and peptides into surgically isolated and continuously perfused segments of rat small intestine. Up to nine discrete antimicrobial molecules appeared in the intestinal perfusates following intravenous administration of bethanechol, a cholinergic agonist, or intralumenal instillation of lipopolysaccharide (LPS). Among them were three markers of Paneth cell secretion: lysozyme; type II (secretory) phospholipase A2; and at least one intestinal defensin, RIP-3, that appeared to be an alternatively processed variant of the rat neutrophil defensin RatNP-3. Both bethanechol- and LPS-stimulated intestinal lumenal perfusates (washings) contained molecules that killed Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes in vitro. These molecules were more active against the avirulent S. typhimurium strain 7953S (phoP) than against its virulent parent, S. typhimurium 14028S. These data demonstrate that small intestinal Paneth cells secrete antimicrobial peptides in vivo, that this secretion is regulated by the autonomic (parasympathetic) cholinergic nervous system, and that the release of antimicrobial molecules can be triggered by the presence of bacterial LPS in the intestinal lumen.

Amino Acid Sequence↗