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J H Walsh

Publications and source records attributed to J H Walsh.

At least 37 records · Page 2Linked to original sources
PubMed2000

Role of PACAP1 receptor in regulation of ECL cells and gastric acid secretion by pituitary adenylate cyclase activating peptide.

We previously reported that PAC1 is expressed on ECL cells resulting in stimulation of [Ca2+]i, histamine and acid secretion. The study reported here characterized the signaling by PAC1 on ECL cells; determined the effects of PACAP on the gastric acid secretion in vivo, and determined the effects of chronic administration of PACAP-27 on ECL cell proliferation. PACAP-27 dose dependently stimulated ECL cell Ca2+ and AC with detectable stimulation at 1 nM and maximal stimulation at 100 nM (six-fold). In rats PACAP-27 administration (10 pmol/kg/h) increased the rate of gastric acid secretion when an antisomatostatin antibody was co-administered. Chronic administration of PACAP (10 pmol/h for seven days) via osmotic pump resulted in a more than twofold increase in BrdU incorporation into ECL cells. PACAP acting at the PAC1 results in dual signaling responses to both [Ca2+]i. AC in ECL cells stimulates gastric acid secretion via the actions of histamine acting at the parietal cell and in whole animals leads to proliferation of ECL cells when administered chronically.

Adenylyl Cyclases↗
PubMed2000

Requirement of cortical actin organization for bombesin, endothelin, and EGF receptor internalization.

The role of actin organization in occupancy-induced receptor internalization remains poorly defined. Here we report that treatment of mouse Swiss 3T3 cells with latrunculin A, a potent inhibitor of actin polymerization (including cortical actin), inhibited the internalization of the endogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judged by uptake of (125)I-labeled GRP or fluorescent Cy3-labeled bombesin. In contrast, cells pretreated with cytochalasin D showed minimal inhibition of bombesin/GRP receptor internalization. Similarly, pretreatment of Swiss 3T3 cells with the potent Rho-kinase inhibitor HA-1077, at concentrations (10-20 microM) that abrogated bombesin-mediated stress fiber formation, did not significantly alter receptor-mediated internalization of (125)I-GRP. These results indicate that bombesin/GRP receptor internalization depends on latrunculin A-sensitive cortical actin rather than on rapidly turning over actin stress fibers that are disrupted by either cytochalasin D or HA-1077. The rates and total levels of internalization of the endogenously expressed endothelin A receptor and epidermal growth factor receptor were also markedly reduced by latrunculin A in Swiss 3T3 cells. The potency of latrunculin A for inhibiting G protein-coupled receptor endocytosis was comparable to that for reducing internalization of the epidermal growth factor tyrosine kinase receptor. We conclude that cortical actin structures, disrupted by latrunculin A, are necessary for occupancy-induced receptor internalization in animal cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Primary structures of PYY, [Pro(34)]PYY, and PYY-(3-36) confer different conformations and receptor selectivity.

We synthesized PYY-(1-36) (nonselective between Y(1) and Y(2) receptor subtype agonists), [Pro(34)]PYY (selective for Y(1)), and PYY-(3-36) (selective for Y(2)) to determine whether solution conformation plays a role in receptor subtype selectivity. The three peptides exhibited the expected specificities in displacing labeled PYY-(1-36) from cells transfected with Y(1) receptors (dissociation constants = 0.42, 0.21, and 1,050 nM, respectively) and from cells transfected with Y(2) receptors (dissociation constants = 0.03, 710, and 0.11 nM, respectively) for PYY-(1-36), [Pro(34)]PYY, and PYY-(3-36). Sedimentation equilibrium analyses revealed that the three PYY analogs were 80-90% monomer at the concentrations used for the subsequent circular dichroism (CD) and (1)H-nuclear magnetic resonance (NMR) studies. CD analysis measured helicities for PYY-(1-36), [Pro(34)]PYY, and PYY-(3-36) of 42%, 31%, and 24%, suggesting distinct differences in secondary structure. The backbone (1)H-NMR resonances of the three peptides further substantiated marked conformational differences. These patterns support the hypothesis that Y(1) and Y(2) receptor subtype binding affinities depend on the secondary and tertiary solution state structures of PYY and its analogs.

Amino Acid Sequence↗
PubMed2000

Pancreatic CCK(B) receptors: their potential roles in somatostatin release and delta-cell proliferation.

In rodents, cholecystokinin (CCK) induces pancreatic enzyme secretion and pancreas growth through its CCK(A) receptors. It is unknown whether occupation of the CCK(B) receptors present in pig and human pancreas can cause the same effects. This study evaluates CCK(B) receptor expression in rat, mouse, pig, and fetal human pancreata using Northern blot, Western blot, and immunofluorescence techniques. The reported 2.7-kb CCK(B) receptor mRNA transcript in the rat brain and gastric fundus is absent in pancreas; the message was, however, detected by RT-PCR and by a CCK(B) receptor antibody as an 80-kDa protein present uniquely in islet delta-cells. Proteins of 50 and 80 kDa appear in mouse pancreas, and proteins of 50 and 115 kDa appear in pig and human pancreas, respectively, all localized in islet delta-cells. Gastrin mRNAs are strongly present in fetal rat pancreas, and the hormone is localized in islets; both are repressed 10 days after birth. In conclusion, the CCK(B) receptors are present in pancreas of four species with exclusive location in islet delta-cells. In such a location, they could be indirectly involved in the control of enzyme secretion.

Animals↗
PubMed1999

Galpha(13) stimulates Rho-dependent activation of the cyclooxygenase-2 promoter.

Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells. Here, we examine the regulation of COX-2 promoter activity by alpha subunits of heterotrimeric G proteins in NIH 3T3 cells. Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for alpha subunits of G-proteins, we show that overexpression of wild type and constitutively active Galpha(13) and Galpha(q) induced transcription from the COX-2 promoter. The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active Galpha(13)(Q226L). We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter. Co-expression of Clostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either Galpha(13)Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin. We conclude that Galpha(13) signals through a Rho-dependent pathway leading to activation of the COX-2 promoter. This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by Galpha(13)/Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.

3T3 Cells↗
PubMed1999

Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor.

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and beta-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of beta-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or beta-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of beta-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and beta-arrestin1 into the same endosomes containing SP and the transferrin receptor (2-10 min); (c) prolonged colocalization of the NK1R and beta-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and beta-arrestin1 in the cytosol (4-6 h). SP stimulated a similar redistribution of immunoreactive beta-arrestin1 and beta-arrestin2. In contrast, SP did not affect Galphaq/11 distribution, which remained at the plasma membrane. Expression of the dominant negative beta-arrestin319-418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of beta-arrestins to the plasma membrane, where they participate in NK1R endocytosis. beta-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and beta-arrestins return to the cytosol.

Animals↗
PubMed1999

An immunohistochemical marker for Wallerian degeneration of fibers in the central and peripheral nervous system.

This work was prompted by the accidental observation that a newly developed, affinity purified polyclonal antibody against the C-terminus of the neuropeptide tyrosine (NPY) Y1-receptor protein decorates degenerating fibers in the central nervous system (CNS). This staining did not appear in control animals in which the antibody marked perikarya and dendrites at previously described locations [X. Zhang, L. Bao, Z.-Q. Xu, J. Kopp, U. Arvidsson, R. Elde, T. Hökfelt, Localization of neuropeptide Y Y1-receptors in the rat nervous system with special reference to somatic receptors on small dorsal root ganglion neurons, Proc. Natl. Acad. Sci. USA 91 (1994) 11738-11742]. Three models of experimental lesions were studied: sciatic nerve transection, spinal cord transection and parietal cortex thermocoagulation. In each model, animals were divided in groups (n=2) and processed for indirect immunofluorescence at different time intervals up to 28 days post-lesion (PL) (see below). All three experimental lesions produced a very intense immunolabeling of fibers in the projection pathways of the lesioned structures, strongly reminding of Wallerian degeneration (WD). In the sciatic nerve, the staining first appeared on day 1 PL, was strongly increased on day 3 PL, then declined after 7 days and had almost completely disappeared after 14 days. In the CNS, the staining appeared later and was first observed on day 3 PL and remained for a longer period, thus showing different time courses in the brain and spinal cord as compared to the sciatic nerve. The labeling was completely abolished, both in the CNS and in the sciatic nerve, by pre-incubation of the Y1-R antibody with the immunogenic peptide at a dilution of 10-6 M. The appearance of the staining and its time course strongly suggest that the process was related to degenerating axons. Although the protein actually detected remains to be determined, it is suggested that the staining ability of this antibody could be used as a positive marker of axonal degeneration following experimental or naturally occurring lesions of the nervous system.

Animals↗
PubMed1999

PYY immunoneutralization does not alter lipid-induced inhibition of gastric emptying in rats.

UNLABELLED: PYY is released from the distal ileum by fat and may be involved in mediating lipid-induced inhibition of gastric acid secretion and intestinal motility. The role of PYY in intestinal lipid-induced inhibition of gastric emptying in awake rats was investigated using a specific polyclonal antibody raised against PYY. METHODS: Gastric emptying of liquids was measured in awake rats fitted with a Thomas gastric cannula. Intralipid (total dose 50 or 100 mg) was perfused for 10 min (0.05 ml/min) into a duodenal (n = 11) or mid-intestinal cannula (60 cm from Ligament of Treitz; n = 8), and gastric emptying was measured over the 5-10 min period. Gastric emptying was measured 15 min after IP injection of PYY (1 nmol/rat). PYY antibody (20 mg) or a control antibody (anti-KLH; keyhole limpet hemocyanin) was injected ip 8-12 h before experiments. RESULTS: Exogenous PYY (1 nmol) inhibited gastric emptying and administration of PYY antibody blocked this response. Perfusion of lipid (50 and 100 mg) into the proximal intestine produced a 46% and 66% inhibition of gastric emptying respectively. Inhibition of gastric emptying in response to 50 mg lipid in the proximal small intestine was unaffected by administration of PYY antibody but was abolished by administration of the CCK A receptor antagonist devazepide (0.1 mg/kg ip). Perfusion of lipid into the distal intestine (50 and 100 mg) inhibited gastric emptying by 10% and 32% respectively. Inhibition of gastric emptying in response to 100 mg lipid in the distal intestine was unaffected by PYY antibody. CONCLUSIONS: Lipid perfused into either the proximal or distal intestine inhibits gastric emptying via a PYY-independent mechanism. CCK is involved in proximal lipid induced inhibition of gastric emptying.

Animals↗
PubMed1999

Expression of cholecystokinin A receptors in neurons innervating the rat stomach and intestine.

BACKGROUND & AIMS: Two distinct receptors, cholecystokinin (CCK)-A and CCK-B, mediate CCK effects in the digestive system. The aim of this study was to elucidate the cellular sites of expression of CCK-A receptor in the rat stomach and small intestine. METHODS: We developed and characterized an antibody to the N-terminal region (LDQPQPSKEWQSA) of rat CCK-A receptor and used it for localization studies with immunohistochemistry. RESULTS: Specificity of the antiserum was demonstrated by (1) detection of a broad band at 85-95 kilodaltons in Western blots of membranes from CCK-A receptor CHO-transfected cells; (2) cell surface staining of CCK-A receptor-transfected cells, (3) translocation of CCK-A receptor immunostaining in CCK-A receptor-transfected cells after exposure to CCK; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with the peptide used for immunization. CCK-A receptor immunoreactivity was localized to myenteric neurons and to fibers in the muscle and mucosa. In the stomach, myenteric neurons and mucosal fibers were abundant. Many CCK-A receptor myenteric neurons contained the inhibitory transmitter vasoactive intestinal polypeptide, and some were immunoreactive for the excitatory transmitter substance P. Subdiaphragmatic vagotomy reduced the density of CCK-A receptor fibers in the gastric mucosa by approximately 50%, whereas celiac/superior mesenteric ganglionectomy had no detectable effect on fiber density. CONCLUSIONS: CCK-A receptor is expressed in functionally distinct neurons of the gastrointestinal tract. CCK-A receptor may mediate reflexes stimulated by CCK through the release of other transmitters from neurons bearing the receptor.

Amino Acid Sequence↗
PubMed1999

Urethane-induced somatostatin mediated inhibition of gastric acid: reversal by the somatostatin 2 receptor antagonist, PRL-2903.

Urethane increases the release of somatostatin (SRIF) which inhibits gastric acid secretion. The SRIF monoclonal antibody, CURE.S6 and the novel sst2 antagonist, PRL-2903 injected intravenously at maximal effective doses increased gastric acid secretion by 2 and 10 fold respectively from basal values within 30 min in urethane-anesthetized rats. Plasma gastrin levels were elevated 2.5 fold within 15 min by PRL-2903 (1.3 micromol/kg, i.v.). These data indicate that the low gastrin and acid secretion levels induced by urethane result from endogenous SRIF acting on sst2 and that PRL-2903 is a valuable SRIF antagonist to assess sst2 mediated events.

Anesthetics, Intravenous↗
PubMed1999

Somatostatin receptor subtype 2A expression in the rat retina.

Somatostatin is mainly expressed by sparsely occurring amacrine and interplexiform cells in the retina. In this study, we characterized the expression and cellular localization of one of the somatostatin subtype (sst) receptors, sst2A, in the rat retina. The presence of sst2A receptor messenger RNA in retinal extracts was demonstrated by reverse transcription-polymerase chain reaction using specific primers to detect the sst2 receptor and its isoforms, sst2A and sst2B. Specific sst2A receptor immunoreactivity was mainly localized to the plasma membrane of several neuronal cell types. In the outer retina, immunoreactivity was localized to cone photoreceptors, horizontal cells, and rod and cone bipolar cells. Double-label experiments showed the co-localization of sst2A receptor and protein kinase C (alpha and beta), a rod bipolar cell marker, and of sst2A receptor and Calbindin-D28k, a horizontal cell marker. In the inner retina, sst2A receptor immunoreactivity occurred in tyrosine hydroxylase-positive amacrine cells; most were of medium to large size. These findings indicate that somatostatin may act at a distance, in a paracrine manner, on several cell types that express the sst2A receptor, and therefore exert a broad modulatory influence on both scotopic and photopic visual pathways.

Animals↗
PubMed1999

High prevalence of cagA-positive strains in Helicobacter pylori-infected, healthy, young Chinese adults.

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.

Adolescent↗
PubMed1999

Differential inhibition of insulin and islet amyloid polypeptide secretion by intraislet somatostatin in the isolated perfused human pancreas.

Islet amyloid polypeptide (IAPP) and insulin are co-stored and generally secreted in parallel; however, studies have demonstrated that the IAPP/insulin molar secretory ratio may be altered in response to certain stimuli. Because we previously demonstrated that intraislet somatostatin is an inhibitory regulator of basal insulin secretion in the isolated perfused human pancreas, this study was designed to determine the relative influence on the regulation of IAPP versus insulin secretion. Single-pass perfusion was performed in pancreata obtained from cadaveric organ donors with continuous perfusion of a modified Krebs media with the glucose level maintained at constant 3.9 mM. Intraislet somatostatin was immunoneutralized by the infusion of either a highly sensitive monoclonal somatostatin antibody (SAb) or its FAb fragment (SFAb). Sequential test periods separated by basal periods were performed by infusion of either of the following: glucose, SAb, SFAb, or appropriate controls. IAPP/insulin molar secretory ratio decreased by 33% in response to infusion of either SAb or the SFAb, respectively (p < 0.01), and decreased by 67% in response to glucose infusion (p < 0.01). An alteration of the IAPP/insulin secretory ratio is seen in response to infusion of exogenous glucose or in response to the neutralization of intraislet somatostatin.

Adolescent↗
PubMed1999

Noninvasive evaluation of Helicobacter pylori therapy: role of fasting or postprandial gastrin, pepsinogen I, pepsinogen II, or serum IgG antibodies.

OBJECTIVE: We evaluated the potential value of a change in serum IgG antibodies, fasting or meal-stimulated gastrin levels, and pepsinogen I (PGI) or pepsinogen II (PGII) levels for identifying Helicobacter pylori (H. pylori) status after antibiotic therapy. METHODS: A total of 32 men and one woman with peptic ulcer disease and documented H. pylori infection were enrolled. Fasting and 30-min postprandial blood samples were obtained at 0, 2, 7, 11, 17, 23, 27, and 39 wk of the study and were analyzed for the factors evaluated. RESULTS: Treatment was successful in 25 patients and failed in seven. Serum IgG antibodies, meal-stimulated gastrin, and both fasting and meal-stimulated pepsinogen I and II levels fell throughout the study, and pepsinogen I:II ratios increased in those whose infection was cured. The mean levels at wk 0 versus wk 7 were: fasting gastrin (fmol/ml) 12.4 and 11, meal-stimulated gastrin 26.5 and 15.4, PGI (ng/ml) 83.7 and 59, PGII (ng/ml) 24.5 and 13.6, PGI/PGII 3.5 and 4.7, and enzyme-linked immunosorbent assay value 4.8 and 4.55. The sensitivity, specificity, and positive and negative predictive values for the data analyzed using different percent changes (e.g., 80%, 50%, and 20%) were calculated. The specificity and sensitivity remained <80% at all time points. CONCLUSIONS: Despite a significant fall in serum markers of H. pylori infection in groups of individuals, no marker tested could be used to reliably determine posttherapy H. pylori status for individual patients.

Adult↗
PubMed1999

Inhibition of pentagastrin-induced gastric acid secretion by intravenous pantoprazole: a dose-response study.

OBJECTIVE: The purpose of this study was to compare the gastric acid inhibitory ability of increasing doses of intravenous (i.i.) pantoprazole with that of i.v. famotidine and placebo. Pentagastrin was infused continuously in healthy subjects as a model for patients with Zollinger-Ellison syndrome. METHODS: Pentagastrin (1 microg/kg/h) was infused to stimulate maximum acid output in 39 subjects over a 25-h period. After 60 min of pentagastrin infusion, subjects received a single dose of i.v. pantoprazole (20, 40, 80, or 120 mg), i.v. famotidine (20 mg), or saline placebo. The variables measured were onset of response (time until acid output fell to < 10 mEq/h), duration of response (time acid output remained < 10 mEq/h), and cumulative acid output over 24 h. RESULTS: All doses of i.v. pantoprazole produced a dose-dependent suppression of acid output to < 10 mEq/h. Single i.v. doses of pantoprazole, 80 and 120 mg, suppressed acid output by > 90% in all subjects for < or = 21 h and had an onset of action of < 1 h. CONCLUSIONS: Intravenous pantoprazole has a rapid onset and a clear dose-related effect, with a significantly longer duration of action than that of i.v. famotidine.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1999

Intestinal fat-induced inhibition of meal-stimulated gastric acid secretion depends on CCK but not peptide YY.

Fat in small intestine decreases meal-stimulated gastric acid secretion and slows gastric emptying. CCK is a mediator of this inhibitory effect (an enterogastrone). Because intravenously administered peptide YY (PYY) inhibits acid secretion, endogenous PYY released by fat may also be an enterogastrone. Four dogs were equipped with gastric, duodenal, and midgut fistulas. PYY antibody (anti-PYY) at a dose of 0.5 mg/kg or CCK-A receptor antagonist (devazepide) at a dose of 0.1 mg/kg was administered alone or in combination 10 min before the proximal half of the gut was perfused with 60 mM oleate or buffer. Acid secretion and gastric emptying were measured. We found that 1) peptone-induced gastric acid secretion was inhibited by intestinal fat (P < 0.0001), 2) inhibition of acid secretion by intestinal fat was reversed by CCK-A receptor antagonist (P < 0.0001) but not by anti-PYY, and 3) slowing of gastric emptying by fat was reversed by CCK-A antagonist (P < 0. 05) but not by anti-PYY. We concluded that inhibition of peptone meal-induced gastric acid secretion and slowing of gastric emptying by intestinal fat depended on CCK but not on circulating PYY.

Animals↗
PubMed1999

COOH-terminally extended secretins are potent stimulants of pancreatic secretion.

Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and alpha-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 +/- 9% (P > 0.05) and 176 +/- 13% (P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.

Amino Acid Sequence↗