Gastrin-like Immunoreactivity Research Articles

Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs

The presence and distribution of cells positive to orexin A (OXA) and to orexin type 2 receptor (OX2R) were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

Diabetic mastopathy mimicking neoplastic lesion — a case report

Ex vivo hemoperfused pancreaticoduodenal preparations from dogs have been used to study intraluminal and circulatory patterns of release of immunoreactive gastrin under basal conditions and after secretin stimulation. Bidirectional release of immunoreactive gastrin was maximal at 3 U/min secretin infusion, and release into pancreatic and duodenal juice exceeded that into portal venous blood. Molecular sieving chromatography of peptides with gastrin-like immunoreactivity recovered from duodenal and pancreatic juice indicated a single species of a molecular size equivalent to CCK8 and smaller than minigastrin (G-14). The exact identity has not been defined. This study demonstrates that secretin stimulates release of gastrin-like peptides into blood and lumen of extra-antral gastrin-producing tissues in the dog. Unidirectional gastrin release patterns from gastrinoma tissue may explain the paradoxical increase in plasma gastrin levels in response to secretin in patients with gastrinomas (Zollinger-Ellison syndrome).

Cimetidine in painful bladder syndrome: a histopathological study.

To determine whether oral cimetidine, which reportedly improves symptoms in 60-70% of patients with painful bladder syndrome/interstitial cystitis has a similar mechanism of action on the human bladder and involving a similar peptidergic pathway as it has in human stomach, where it alters histamine-gastrin reactions mediated via H2 receptors and a proton pump. Fourteen patients (13 women and one man, mean age 51 years) with refractory bladder pain and irritative urinary symptoms were treated with cimetidine. The response to cimetidine was assessed by outpatient follow-up and an analogue pain score. Bladder biopsies from eight patients were stained with haematoxylin and eosin, and a polyclonal antibody to gastrin, with counterstaining using toluidine blue, to detect mast cell granules containing histamine. Biopsies from normal areas of the bladder from an age-matched control group of patients with transitional cell carcinoma of the bladder were stained similarly. Human stomach and colon were used as positive controls for gastrin and toluidine blue, respectively. The clinical response to cimetidine was compared with the histology in a blinded fashion. Eight of the patients responded well to cimetidine; none of the biopsies showed evidence of carcinoma in situ. Although G cells in the stomach stained well for gastrin none of the bladder biopsies showed gastrin-like immunoreactivity, apart from some nonspecific urothelial staining. Numerous mast cells with crimson granules and pale nuclei were visible in three patients and fewer in three others; their presence or absence did not correlate with the symptomatic response. Cimetidine is a useful medical treatment for bladder pain but the presence or absence of gastrin or histamine-like immunoreactivity does not explain its therapeutic benefit.

Heterogeneity of cholecystokinin/gastrin-like immunoreactivity in the nervous system of the nematodeAscaris suum

A wholemount immunocytochemical method was used for the localization of cholecystokinin (CCK8)-like and gastrin-like immunoreactivity in Ascaris. The patterns of specific neuronal staining given by two antisera and four monoclonal antibodies made against CCK8, and one antiserum made against gastrin were investigated. Preabsorption of these antibodies with CCK8 or gastrin 17 resulted in complete loss of immunoreactivity in almost all of the neurons (two antisera also contained nonspecific antibodies), suggesting that all of the antibodies recognize epitopes, in Ascaris neurons, that include some or all of the C-terminal five amino acids that are identical in CCK8 and gastrin 17. However, the seven different antibodies showed immunoreactivity in different subpopulations of neurons, implying that there are at least seven different species of CCK-like molecules in Ascaris. Fractionation of Ascaris peptide extracts by high performance liquid chromatography (HPLC), monitoring fractions with a CCK8 radioimmunoassay (RIA), also shows heterogeneity of molecules immunologically related to CCK8.

Heterogeneity of cholecystokinin/gastrin‐like immunoreactivity in the nervous system of the nematode Ascaris suum

A wholemount immunocytochemical method was used for the localization of cholecystokinin (CCK8)-like and gastrin-like immunoreactivity in Ascaris. The patterns of specific neuronal staining given by two antisera and four monoclonal antibodies made against CCK8, and one antiserum made against gastrin were investigated. Preabsorption of these antibodies with CCK8 or gastrin 17 resulted in complete loss of immunoreactivity in almost all of the neurons (two antisera also contained nonspecific antibodies), suggesting that all of the antibodies recognize epitopes, in Ascaris neurons, that include some or all of the C-terminal five amino acids that are identical in CCK8 and gastrin 17. However, the seven different antibodies showed immunoreactivity in different subpopulations of neurons, implying that there are at least seven different species of CCK-like molecules in Ascaris. Fractionation of Ascaris peptide extracts by high performance liquid chromatography (HPLC), monitoring fractions with a CCK8 radioimmunoassay (RIA), also shows heterogeneity of molecules immunologically related to CCK8. © 1996 Wiley-Liss, Inc.

Change in subcellular localization of gastrin-like immunoreactivity in epithelial cells of rat duodenum induced by carbachol.

The present study provides morphological evidence to support the contention of exocrine secretion from duodenal gastrin-containing cells. The isolated vascularly perfused duodenal preparation with or without carbachol stimulation was used. At the end of perfusion, tissue was fixed and prepared for electron microscopic examination. For immunoelectron microscopic study for gastrin, postembedding immunogold reaction combined with preembedding DAB staining was used. In saline-treated controls, DAB reaction was restricted to the basal cytoplasm and immunogold labeling was concentrated over electron-dense cores of secretory granules packed at the basal cytoplasm. However, in carbachol-stimulated animals, immunogold labeling as well as DAB reactions were accumulated at the apical portion of the cytoplasm, suggesting that a high concentration of gastrin was involved in the apical cytoplasm. In carbachol-stimulated cells, aggregation of small vesicles was observed beneath the microvilli, and most of these vesicles had no cores but were similar in size to the basal secretory granules. Immunogold particles were diffusely scattered at the cytoplasm outside these vesicles. These findings suggest that the gastrin-like immunoreactivity was pooled at the matrix of apical cytoplasm in carbachol-stimulated cells, which might be derived from the secretory granules migrated from the basal cytoplasm into apical portion of the cells. In conclusion, the present study demonstrated the change in subcellular localization of gastrin-like immunoreactivity in intestinal gastrin cells after stimulation with carbachol. Aggregation of immunoreactivity at the apical portion of the cells suggests that gastrin may be released into the intestinal lumen.

Low tissue gastrin content in the porcine distal duodenum is associated with increased percentage of G34

In the gastric antrum of pigs aged between 2 and 171 days, G17 accounted for most of the gastrin-like immunoreactivity and G34 for up to 2%. G34 increased progressively from a mean of 7% in the duodenal bulb to 39% in the most distal segment of the duodenum. In both the proximal duodenum and for the whole duodenum, the percentage of G34 correlated negatively with the tissue gastrin content. The degree of sulphation was similar in the antrum and proximal duodenum (mean 67% G17 non-sulphated).

CCK/Gastrin-like Immunoreactivity in the Goldfish Pituitary: Regulation of Pituitary Hormone Secretion by CCK-like Peptides in Vitro

The presence and distribution of cholecystokinin (CCK)/gastrin-like immunoreactivity (IR) was examined in the goldfish pituitary. Intense CCK/gastrin-like IR was consistently observed within fibers of the proximal pars distalis (PD), with fewer IR fibers localized in the rostal PD. Within the proximal PD CCK/gastrin-like IR fibers were distributed among both the gonadotrophs and somatotrophs, suggesting a possible role for CCK-like peptides in the regulation of gonadotropin-II (GtH-II) and growth hormone (GH) secretion. Exposure of pituitary fragments from either sexually gonadal recrudescing (maturing) or regressed goldfish to three 5-min pulses of 1.0 or 10 nM sulfated CCK8 (CCK8-s), at 55-min interpulse intervals, resulted in an increased secretion of both GtH-II and GH. Independent of the dose perifused, the GtH-II release responses to the second and third pulses of CCK8-s were always of similar magnitude to the first pulse at a given dose. For GH, repeated challenges of pituitary fragments to 0.1 or 1.0 nM CCK8-s stimulated release responses of similar magnitude; however, successive pulses of 10 nM CCK8-s resulted in a desensitization in the GH release response to the second or the third pulse. Fragments from sexually regressed goldfish exhibited an overall greater release response of GtH-II to CCK8-s relative to fragments from sexually recrudescing fish, whereas the GH release responses to CCK8-s were similar between the two sexual stages. A dose-dependent release of GtH-II was present in pituitary fragments from sexually regressed goldfish following five 5-min pulses of increasing (0.1 to 100 nM) doses of CCK8-s. Finally, the sulfated forms of CCK8 and gastrin 17 (G17-s) exhibited greater stimulatory abilities than the nonsulfated form of CCK8 in releasing GtH-II and GH from fragments of sexually recrudescing fish. Additionally, CCK8-s and G17-s were equal in their capacity to stimulate the release of GtH-II, whereas G17-s was slightly more effective than CCK8-s in stimulating the release of GH.Overall, these studies are the first to provide evidence that IR CCK/gastrin-like fibers are codistributed among the gonadotrophs and somatotrophs, and that CCK-like peptides are highly effective in stimulating GtH-II and GH release from the goldfish pituitary.

Immunocytochemical identification of some regulatory peptides (gastrin, gastrin-releasing peptide, neurotensin and vasoactive intestinal polypeptide) in the Harderian gland of the green frog, Rana esculenta.

The presence and distribution of gastrin-, gastrin-releasing peptide-, neurotensin- and vasoactive intestinal polypeptide-like immunoreactivity in the Harderian gland of Rana esculenta were studied at different times of the annual cycle. Gastrin-releasing peptide, neurotensin and vasoactive intestinal polypeptide-like substances were found either in the glandular cells, or in the nerve fibers surrounding the glandular acini. Gastrin-like immunoreactivity was confined to the glandular cells. The immunoreactivity varied during the annual cycle, with the greatest concentration being noted during the recovery phase of glandular secretory activity.

Philosophy of pharmacokinetic modelling

Ex vivo hemoperfused pancreaticoduodenal preparations from dogs have been used to study intraluminal and circulatory patterns of release of immunoreactive gastrin under basal conditions and after secretin stimulation. Bidirectional release of immunoreactive gastrin was maximal at 3 U/min secretin infusion, and release into pancreatic and duodenal juice exceeded that into portal venous blood. Molecular sieving chromatography of peptides with gastrin-like immunoreactivity recovered from duodenal and pancreatic juice indicated a single species of a molecular size equivalent to CCK8 and smaller than minigastrin (G-14). The exact identity has not been defined. This study demonstrates that secretin stimulates release of gastrin-like peptides into blood and lumen of extra-antral gastrin-producing tissues in the dog. Unidirectional gastrin release patterns from gastrinoma tissue may explain the paradoxical increase in plasma gastrin levels in response to secretin in patients with gastrinomas (Zollinger-Ellison syndrome).

Precorneal pharmacokinetics

Ex vivo hemoperfused pancreaticoduodenal preparations from dogs have been used to study intraluminal and circulatory patterns of release of immunoreactive gastrin under basal conditions and after secretin stimulation. Bidirectional release of immunoreactive gastrin was maximal at 3 U/min secretin infusion, and release into pancreatic and duodenal juice exceeded that into portal venous blood. Molecular sieving chromatography of peptides with gastrin-like immunoreactivity recovered from duodenal and pancreatic juice indicated a single species of a molecular size equivalent to CCK8 and smaller than minigastrin (G-14). The exact identity has not been defined. This study demonstrates that secretin stimulates release of gastrin-like peptides into blood and lumen of extra-antral gastrin-producing tissues in the dog. Unidirectional gastrin release patterns from gastrinoma tissue may explain the paradoxical increase in plasma gastrin levels in response to secretin in patients with gastrinomas (Zollinger-Ellison syndrome).

Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells.

In previous studies we have shown that the gene encoding cholecystokinin (CCK) is expressed in spermatogenic cells of several mammalian species. In the present study we show that a gene homologous to the CCK-related hormone, gastrin, is expressed in the human testis. The mRNA hybridizing to a human gastrin cDNA probe in the human testis was of the same size (0.7 kb) as gastrin mRNA in the human antrum. By in situ hybridization the gastrinlike mRNA was localized to seminiferous tubules. Immunocytochemical staining of human testis revealed gastrinlike peptides in the seminiferous tubules primarily at a position corresponding to spermatids and spermatozoa. In ejaculated spermatozoa gastrinlike immunoreactivity was localized to the acrosome. Acrosomal localization could also be shown in spermatids with electron microscopy. Extracts of the human testis contained significant amounts of progastrin, but no bioactive amidated gastrins. In contrast, ejaculated sperm contained mature carboxyamidated gastrin 34 and gastrin 17. The concentration of gastrin in ejaculated human spermatozoa varied considerably between individuals. We suggest that amidated gastrin (in humans) and CCK (in other mammals) are released during the acrosome reaction and that they may be important for fertilization.

Molecular variants of cholecystokinin after endogenous stimulation in humans: a time study

The time-dependent release of molecular variants of cholecystokinin (CCK) into the circulation was studied before and 1, 2, and 4 h after a test meal in six healthy volunteers. At each time period, 100 ml of blood were drawn in a manner to inhibit CCK degradation. Plasma was formed and CCK concentrated by Sep-Pak C18 cartridge chromatography. Molecular variants of CCK and gastrin were well separated from each other by high-performance liquid chromatography (HPLC). Molecular forms of CCK and gastrin were measured by radioimmunoassay using an antibody that requires the presence of the carboxyl-terminal phenylalanine amide for full recognition, implying that biologically active forms were detected. HPLC elution positions of gastrin forms were determined using a gastrin-specific antibody. Chromatographic separation of CCK from gastrin forms was complete, allowing separate integration of gastrin and CCK forms. Therefore no subtraction of gastrin-like immunoreactivity from CCK-like immunoreactivity (CCK-LI) was necessary and CCK-LI could be directly determined. Peaks of CCK-LI were integrated in the column eluates and the plasma concentrations were calculated. Total plasma CCK-LI rose from a value of 2.4 +/- 0.6 pM before the test meal to 6.4 +/- 0.8, 6.6 +/- 0.9, and 5.8 +/- 1.2 pM 1, 2, and 4 h postprandially. The major molecular forms released into the circulation eluted on HPLC in the position of CCK-58 and CCK-39 (which coelutes with CCK-33). Minor amounts were detected in the position of CCK-8. There was no significant difference in the relative proportions of the molecular forms released at the different time periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunostaining of antral gastrin cells is quantitatively increased in Helicobacter pylori gastritis

The amount of gastrin-like immunostaining in gastrin (G) cells of the antral mucosa was quantified using a computer-assisted method of measuring immunoreaction product. Biopsies from 25 patients without Heliobacter-like organisms and 60 patients with varying degrees of infection were immunostained for gastrin. Twenty-five G cells from each patient were measured both subjectively and by image analysis. Gastrin-like immunoreactivity was found to be significantly increased in the presence of Heliobacter-like organisms.

Influence of diet on the development of antral gastrin-like immunoreactivity in the stomach of rats.

The effects of individual food constituents on antral gastrin-like immunoreactivity concentrations were studied in young rats. Rats aged 7 to 20 days were given only rat breast milk and then weaned by various nutrients (regular laboratory chow, protein (ovalbumin)-, fat- or carbohydrate (starch)-rich food). Rats receiving rat breast milk only until 27 days of age were also studied. In rats on regular laboratory chow, antral gastrin-like immunoreactivity increased and reached adult levels on day 25. In rats on ovalbumin, fat-rich food or starch, it increased on day 23 but dropped thereafter. The increment by laboratory chow was higher than that by the individual nutrients. No increase was observed during milk feeding alone. Gel filtration of antral gastrin-like immunoreactivity from 25-day-old rats on laboratory chow or three essential nutrients showed the same results.

Isolation and characterization of rabbit gastrin

The heptadecapeptide form of rabbit gastrin was extracted from 16 rabbit antra and purified by a combination of DEAE Sephadex, C-18 SEP PAK cartridges, fast performance liquid chromatography (FPLC) and reverse phase high pressure liquid chromatography (HPLC) steps. After the HPLC purification, a sharp, single peak of gastrin-like immunoreactivity was detected that had the same absorption to immunoreactivity ratio as human gastrin. An amino terminal pyrrolidone carboxylic acid blocking group was removed by incubation with pyrrolidone carboxylic peptidase. The amino acid analysis, microsequence analysis and mass spectrometry all confirmed the structure of rabbit gastrin being pQGPWLQEEEEAYGWMDFamide. This sequence is identical to human gastrin-17 except for glutamine in position 6 which replaces glutamate in human gastrin. Both sulfated and unsulfated rabbit gastrin-17 were characterized by mass spectrometry.

Localization of immunoreactive gastrin-like cells in the alimentary tract of the ascidian Styela plicata

The occurrence of a gastrin-like immunoreactivity in the alimentary tract of the ascidian Styela plicata has been investigated using immunocytochemical methods. Gastrin-like cells are present only in the gastric epithelium among the cell types responsible for digestion and absorption of food. The physiological role played by the ascidian gastrin-like peptides is discussed together with the evolutionary history of peptides of the gastrin/cholecystokinin family.

Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis

Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.

An immunocytochemical and ultrastructural study of endocrine cells in the gut of a teleost fish, Sparus auratus L

Endocrine cells in the gut of Sparus auratus L. (gilt-head sea bream) have been demonstrated by immunocytochemical and electron microscopic techniques. Cells showing somatostatin and gastrin-like immunoreactivity were found in the depth of the gastric folds and in the upper part of the stomach glands while substance P immunoreactive cells were present between the upper epithelial cells of the gastric folds. Cells showing gastrin, substance P, pancreatic polypeptide, cholecystokinin, and Met-enkephalin immunoreactivity were observed in the intestinal mucosa scattered between epithelial cells. Eight types of endocrine cells were ultrastructurally characterized by the shape, size, and electron density of their respective secretory granules. A tentative correlation between these diverse cell types and those identified by immunocytochemical techniques has been established.

Localization and identification of neurons with cholecystokinin and gastrin-like immunoreactivity in wholemounts of Aplysia ganglia

Immunohistochemical procedures were applied to wholemounts of the central nervous system and posterior intestine of the mollusc, Aplysia californica, to facilitate localization of cells that were immunoreactive to several antisera recognizing various epitopes of the peptides cholecystokinin and gastrin. Only antisera that recognized the carboxyl terminal sequence common to cholecystokinin and gastrin reacted with the Aplysia tissues tested. Intracellular electrophysiological studies of identified postsynaptic targets of immunoreactive neurons in the cerebral ganglia indicated that mammalian forms of gastrin 1–17, several cholecystokinin fragments, and the related peptide, amphibian caerulein, did not mimick the synaptic response mediated by the immunoreactive presynaptic neurons. Combinations of electrophysiological, immunohistochemical, and biochemical studies of several neurons in the buccal ganglia indicated that neurons B7 and B13 were immunoreactive to antisera against cholecystokinin and gastrin and that neuron B13 also contained a concentration of the neurotransmitter acetylcholine as high as in the identified cholinergic buccal neurons, B4 and B5. Several differences in the immunoreactivity of the various antisera were observed. Only one of the antisera was effective in staining neurons in the abdominal ganglia and another antiserum stained subsets of neurons that were immunoreactive to most of the other antisera recognizing the carboxyl terminus common to cholecystokinin and gastrin. The giant serotoninergic metacerebral neurons in Aplysia were not immunoreactive to the cholecystokinin/gastrin antisera even though it has been reported that the homologous neurons in a pulmonate mollusc contain cholecystokinin-like immunoreactivity. These studies demonstrated that there are many neurons with cholecystokinin/gastrin-like immunoreactivity in the Aplysia central and peripheral nervous system and suggested that the peptide may differ from vertebrate forms of cholecystokinin and gastrin. The identification of immunoreactive neurons with known postsynaptic target neurons and buccal neurons with acetylcholine co-localized with a cholecystokinin/gastrin-like peptide will facilitate elucidation of the functions of peptides in the nervous system since the Aplysia preparation is well known to be amenable to multidisciplinary studies.