Immunohistochemistry and morphometry of gastrin cells in the rat pyloric antrum during starvation

Summary The gastrin cells (G cells) in the rat pyloric antrum after 7, 14, 21 and 28 days of starvation were investigated by immunohistochemistry and electron microscopy. In the peroxidase anti-peroxidase method for light microscopy, gastrin immunoreactive cells during starvation markedly decreased in number and size. Quantitative electron microscopy revealed that during starvation the number of electron-lucent granules were greatly reduced, but the number of electron-dense granules increased; the number of intermediate granules were not remarkably changed in G cells. These results may suggest that the synthesis of gastrin and granule maturation were greatly inhibited during long-term starvation.     

Augmented vagal release of antral gastrin by 2-deoxyglucose after fundic vagotomy in dogs.

To study the relation between gastrin released by vagal excitation and the secretion of H+ and pepsin under various conditions, central vagal excitation was induced by 2-deoxyglucose (2DG) in doses of 50, 100, and 200 mg/kg body wt given as a single intravenous injection in seven gastric fistula dogs, three with fundic vagotomy and four with intact vagi. Serum gastrin increased linearly with dose doubling in both groups but was twice as high in the vagotomized dogs. Total acid output for 3 h was related linearly to integrated gastrin output in both groups, but the slope, H+/gastrin, was 10 times steeper in the vagally intact dogs (330 vs. 34 mueq/pg gastrin-ml-30 min) and pepsin output almost 20 times greater [5,400 peptic units (PU) vs. 296 PU]. Acidification of the antrum to pH 1.2-1.4 eliminated the gastrin response to 2DG in both groups of dogs. Atropine (100 microgram/kg iv) reduced serum gastrin in the vagotomized and increased it in the intact dogs. Atropinization uncovers stimulation by 2DG by pathways that do not involve muscarinic cholinergic receptors. Stimulation by both pathways is suppressible by acid. We conclude that fundic vagotomy removes an inhibitor of vagal gastrin release.     

Organ culture studies of nude mouse thymus.

There is evidence that peripheral lymphoid organs of nude mice, born from homozygous matings, contain a small proportion of theta-positive lymphocytes indicating that nude mice may not be totally devoid of T cell function. It has been suggested that such lymphocytes may develop within the dysplastic nude thymus itself. While this suggestion receives no support from morphological studies, it has been claimed that on explanation to organ culture the developing nude thymus becomes lymphoid. In this present study we confirm the presence of theta-positive lymphocytes in peripheral lymphoid tissues of homozygous nude mice born of nude parents. However, when we have organ-cultured nude thymus, explained from homozygous nude embryos at days 13, 14, 16 and 18 of gestation, we have found no histological sign of lymphopoiesis nor have we detected any theta-positive cells in such cultured material. On the contrary, the nude thymus in vitro develops into the polycystic structure characteristic of the adult nude thymus. We conclude that the small number of theta-positive cells present in the periphery result from extrathymic differentiation.     

Effect of intraantral pH on basal gastrin release into the circulation and antral lumen in anesthetized cats

Uvnås -Wallensten , K. Effect of intraantral pH on basal gastrin release into the circulation and antral lumen in anesthetized cats. Acta physiol. scand. 1978. 104. 386–393. In acute experiments on cats antral pouches were perfused with solutions of different pH (1–13). After antrum passage the gastrin levels in the perfusates were measured with radioimmunoassay and the amounts of gastrin released into the antral lumen per minute calculated. The venous gastrin levels were determined concomitantly. Small amounts of gastrin (1 000–1 500 pg/min) were released into the antrum during perfusion with 0.1 M HCI. Subsequent perfusion with 0.15 M NaCl (pH 6.8) did not significantly increase the release of gastrin. On the other hand, 0.1 M phosphate buffer (pH 7.4) caused a dramatic augmentation of the gastrin output into the antral lumen (?17 fold). A concomitant increase of peripheral gastrin levels was observed. Also other alkaline solutions such as 0.15 M NaHCO, (pH 8), 0.15 M Tris buffer (pH 8) or 0.01 and 0.1 M NaOH (pH 12 and 13) promoted the release of gastrin. It is discussed whether the gastrin release at alkaline pH is induced by the alkaline pH itself or by anions such as HPO-₄ HCO-₃ and OH-. The apparent effect of pH could then be due to the formation of these ions at higher pH.     

Organ culture of fetal rat small intestine for testing gluten toxicity: a reappraisal

Jejunal segments from fetal rats of 18 days gestation were maintained in an organ culture system for up to 72 h. During this period, villi developed within the intestinal lumen and the epithelium changed from stratified to simple columnar. Peristaltic activity was observed during in-vitro culture. Alkaline phosphatase specific activity of the bowel segments fell after 48 hours culture, compared with pre-culture values (P less than 0.05), but that of alpha-glucosidase increased. The addition of Frazer's gluten fraction III to the culture medium caused slowing in the rate of morphological maturation of the jejunal explants, but there was no additional effect on enzyme specific activities compared with segments cultured in gluten-free medium. The place of organ culture of fetal rat intestine as an animal model for testing cereal toxicity in the study of coeliac disease remains unclear.     

In vitro gastrin secretion by rat antrum: effects of neurotransmitter agonists, antagonists, and modulators of secretion

Gastrin release was studied from rat antral mucosal cells maintained under tissue culture conditions. Results of experiments with neurotransmitter agonists, antagonists, and modulators indicate the following: Adrenergic mediation of gastrin release occurs in vitro. Adrenergic stimulatory action could be demonstrated by norepinephrine and, more specifically, beta-receptor activators, whereas an alpha-receptor activator was inhibitory. The effects appear to be specific because the agonists stimulate gastrin release at low concentrations and because these effects are blocked by the appropriate antagonists. Because high carbachol concentrations were needed to produce gastrin secretion, and inhibition by atropine occurred also at high concentrations, these effects may be nonspecific. Both carbachol- and norepinephrine-mediated gastrin release are modified by somatostatin and adenosine. Effective modulation of gastrin release by these substances may be relevant in understanding the fine regulation of gastrin release during digestive processes.     

Effect of intraantral pH on basal gastrin release into the circulation and antral lumen in anesthetized cats.

In acute experiments on cats antral pouches were perfused with solutions of different pH (1-13). After antrum passage the gastrin levels in the perfusates were measured with radioimmunoassay and the amounts of gastrin released into the antral lumen per minute calculated. The venous gastrin levels were determined concomitantly. Small amounts of gastrin (1,000--1,500 pg/min) were released into the antrum during perfusion with 0.1 M HCl. Subsequent perfusion with 0.15 M NaCl (pH 6.8) did not significantly increase the release of gastrin. On the other hand, 0.1 M phosphate buffer (pH 7.4) caused a dramatic augmentation of the gastrin output into the antral lumen (approximately 17 fold). A concomitant increase of peripheral gastrin levels was observed. Also other alkaline solutions such as 0.15 M NaHCO3 (pH 8), 0.15 M Tris buffer (pH) or 0.01 and 0.1 M NaOH (pH 12 and 13) promoted the release of gastrin. It is discussed whether the gastrin release at alkaline pH is induced by the alkaline pH itself or by anions such as HPO-4, HCO-3 and OH-. The apparent effect of pH could then be due to the formation of these ions at higher pH.     

Organ culture of fetal rat small intestine for testing gluten toxicity: a reappraisal.

Jejunal segments from fetal rats of 18 days gestation were maintained in an organ culture system for up to 72 h. During this period, villi developed within the intestinal lumen and the epithelium changed from stratified to simple columnar. Peristaltic activity was observed during in-vitro culture. Alkaline phosphatase specific activity of the bowel segments fell after 48 hours culture, compared with pre-culture values (P less than 0.05), but that of alpha-glucosidase increased. The addition of Frazer''s gluten fraction III to the culture medium caused slowing in the rate of morphological maturation of the jejunal explants, but there was no additional effect on enzyme specific activities compared with segments cultured in gluten-free medium. The place of organ culture of fetal rat intestine as an animal model for testing cereal toxicity in the study of coeliac disease remains unclear.     

Staining methods for morphometric studies of parietal and gastrin cells in the rat stomach

In order to select the most suitable procedures for quantitative microscopy of both parietal and gastrin cell populations in the rat stomach, various staining methods were compared. For parietal cell identification in particular, the following procedures were tested: i) the modification of the haematoxylin-eosin method proposed by Marks and Drysdale, ii) the haematoxylin-eosin-saffron fluorochrome stain on paraffin sections, iii) the haematoxylin-azophloxin-saffron fluorochrome stain on paraffin sections, and iv) the May-Grunwald-Giemsa stain on thin sections from plastic-embedded specimens. This last provided the best results in parietal cell individualization and seemed to be the most suitable method for an accurate image analysis. Immunohistochemistry was the only unequivocal way to identify gastrin cells. Two variant procedures were examined; a) the agar-paraffin embedding technique, and b) the combination of a mucin staining with the immunoperoxidase reaction. The first technique provided an easier procedure for handling seriate strips of gastric mucosa for proper enumeration of immunostained cells. The second was presented as a promising variant procedure for a combined investigation of both G-cell population and mucin secretion patterns under differnt experimental conditions in the same specimen.     

Effect of intraluminal pH on the release of somatostatin and gastrin into antral, bulbar and ileal pouches of conscious dogs

Experiments were performed on conscious dogs with chronic pouches of the antrum, the duodenal bulb or the ileum, which were perfused with solutions of varying pH. Gastrin and somatostatin levels were measured in the perfusates. When the pouches were perfused with 0.15 M NaCl only small amounts of gastrin and somatostatin (1 pmol/min) were released into the lumen of the antrum and of the duodenal bulb. By lowering pH of the perfusion fluid a pH dependent release of somatostatin was induced into the lumen of the antrum and the duodenal bulb. Perfusion with 0.1 M HCl caused a large output of somatostatin (6--60 pmol/min) into the pouches. The upper pH limit for stimulation of the intraantral or intrabulbar somatostatin release appeared to be approximately pH 3--4. Somatostatin was also released into ileal perfusates at intraluminal pHs below 3--4. Lowering of pH in the antral pouches caused an increased intraluminal gastrin release, which was quantitatively less impressive than that of somatostatin. Occasionally also the gastrin release into the duodenal bulb increased during perfusion with 0.1 M HCl, whereas no such release was induced by acidification of the lumen of the ileum. It is suggested that the inhibition of gastrin release observed at low intraantral pH is mediated by a local effect of somatostatin, since this peptide is released in a pH dependent manner in the antropyloric region. It is also suggested that acidification of any region of the gastrointestinal tract will stimulate the release of peptides from all endocrine cells of the open type, probably by an unspecific effect on the membrane. Thus both gastrin and somatostatin are released by acidification of the antrum, but in the presence of high local levels of somatostatin, the release of gastrin is substantially inhibited.     

Reduced aspartate release from rat hippocampal synaptosomes loaded with Clostridial toxin light chain by electroporation: evidence for an exocytotic mechanism

Aspartate can be released from certain hippocampal pathways along with glutamate or GABA. Although aspartate immunoreactivity has been localized to synaptic vesicles and aspartate release is Ca(2+)-dependent, there has been no clear evidence favoring an exocytotic mechanism. In particular, pretreatment with Clostridial toxins has not consistently inhibited aspartate release, even when release of glutamate from the same tissue samples was markedly inhibited. To address this issue directly, rat hippocampal synaptosomes were permeabilized transiently by electroporation in the presence of active or inactivated Clostridial toxin light chains. Loading rat hippocampal synaptosomes with the active light chain of tetanus toxin or of botulinum neurotoxins A, B or C reduced the K(+)-evoked release of aspartate at least as much as that of glutamate. These results confirm that aspartate is released by exocytosis in rat hippocampus.     

Organ culture of osteopetrotic (ia) rat bone: Evidence that the defect is cellular

Calvarial bone from osteopetrotic (ia) rats and normal littermates has been cultured in a chemically defined medium supplemented with homologous serum to test for the presence of inhibitors or the absence of promoters of bone resorption in mutant serum. In addition, the response of mutant and normal bone to parathyroid extract and hydrocortisone was tested in vitro. The results indicate that mutant and normal serum do not differ with respect to their ability to support bone resorption and that ia bone responds to hydrocortisone but not parathyroid extract in organ culture. These data indicate that the skeletal defect in ia rats is not humoral but cellular.     

Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence

Release of ³H-ACh from isolated nerve endings of rat hippocampus was evoked by incubation in Krebs-Ringer's buffer containing 25 or 35 mM potassium. The release was Ca²⁺-dependent and could be inhibited by Mg²⁺ (20 mM). The muscarinic antagonist, atropine (10^-10–10^-6 M), enhanced ³H-ACh-release. The muscarinic agonist, carbachol (10^-5–10^-3 M) inhibited ³H-ACh release via interaction with muscarinic receptors: this effect could be blocked by atropine (10^-6 M). The presence of the feed-back regulation of ³H-ACh release in a cell-free preparation provides further evidence that the presynaptic regulation is exerted by muscarinic autoreceptors localized on the cholinergic nerve ending itself. The feed back inhibition of the ³H-ACh release does not require the presence of intact neurons or neuronal loops as tetrodotoxin (2.5. 10^-6 M) does not affect the above results.