Self-replication of somatostatin cells in the antral mucosa of rodents

Abstract. The possibility that antral somatostatin cells have a self-replicating activity has been studied in three species of rodents: mice, rats and guinea-pigs, after a flash tritiated thymidine injection. The immunocytochemical staining of somatostatin cells, using specific antiserum, was combined with radioautographic procedures. The labelling index for somatostatin cells–and for gastrin cells indentified on serial sections–was established after counting a large number of cells at the optical microscope level, on parallel tissue strips removed throughout the entire antrum.
A significant percentage of the somatostatin cell population synthesized DNA. Values were similar for the three species of rodents ranging from 0.8 to 1.1%, that is slightly higher than the percentage of labelled gastrin cells, which was 0.6–0.7%. After a 36-hr continuous infusion of radioactive precursor in one rat, the labelling index observed remained low; 2.33% for somatostatin cells and 1.68% for gastrin cells. Colchicine injection in mice allowed the observation of mitotic figures in well differentiated somatostatin cells. Four hours after that injection, the mitotic index was estimated roughly at 0.3%.
Thus, evidence has been presented that in rodents a fraction of the antral somatostatin cell population is capable of dividing, similar to the situation in gastrin cells.     

Mast and enterochromaffin-like cells of the gastric mucosa]

An attempt has been made to reveal simultaneously both mast and ECL cells in the fundic mucosa of some laboratory animals and man. In Bouin fluid fixed specimens, toluidine blue pH 5.0 and alcian blue pH 1.0 failed to reveal mucosal mast cells in rats and mice only. In those animals mucosal mast cells became demonstrable in Carnoy fluid fixed tissues after staining with alcian blue pH 1.0. A double staining technique has been applied using Grimelius silver method followed by staining either with toluidine blue after acid hydrolysis or with alcian blue. Both mast and ECL cells became visible showing here and there their close arrangement. The latter might be a point for some functional relations between both cell types.     

Self-replication of somatostatin cells in the antral mucosa of rodents

The possibility that antral somatostatin cells have a self-replicating activity has been studied in three species of rodents: mice, rats and guinea-pigs, after a flash tritiated thymidine injection. The immunocytochemical staining of somatostatin cells, using specific antiserum, was combined with radioautographic procedures. The labelling index for somatostatin cells--and for gastrin cells identified on serial sections--was established after counting a large number of cells at the optical microscope level, on parallel tissue strips removed throughout the entire antrum. A significant percentage of the somatostatin cell population synthesized DNA. Values were similar for the three species of rodents ranging from 0.8 to 1.1%, that is slightly higher than the percentage of labelled gastrin cells, which was 0.67-0.7%. After a 36-hr continuous infusion of radioactive precursor in one rat, the labelling index observed remained low; 2.33% for somatostatin cells and 1.68% for gastrin cells. Colchicine injection in mice allowed the observation of mitotic figures in well differentiated somatostatin cells. Four hours after that injection, the mitotic index was estimated roughly at 0.3%. Thus, evidence has been presented that in rodents a fraction of the antral somatostatin cell population is capable of dividing, similar to the situation in gastrin cells.     

Endocrine cells in the antro-pyloric mucosa of the stomach

Summary A type of endocrine-like cell displaying all morphological features of protein secreting cells, has been found in the antro-pyloric mucosa of the stomach of mammals. Light and electron microscopy observations provide a sharp distinction of this cell from the 5-hydroxytryptamine-storing enterochromaffin cell. Its possible involvement in the secretion of a protein or peptide hormone is discussed.This investigation was supported by grant N. 115/1139/991 from the Italian Consiglio Nazionale delle Ricerche.     

Immuno-electron-cytochemical localization of the somatostatin cells in the human antral mucosa.

Immuno-cytochemical methods were used to identify, in light and electron microscopy, the somatostatin-containing cells of the human antral mucosa. By means of immunoperoxidase and immunofluorescence methods sequentially applied on the same section, it was shown that the somatostatin cells are distinct from the gastrin cell population; these two endocrine cell types are often closely related. On ultrathin sections from aldehyde-fixed. Epon-araldite embedded tissues, the site of storage of somatostatin was localized with the peroxidaseantiperoxidase complexes technique, after removal of the resin by means of sodium ethoxide. This procedure represents a new technical approach to the use of electron-cytochemical techniques. The results indicate that somatostatin, a growth hormone release inhibiting factor, is localized in the endocrine granules of the D cells.     

Studies of isolated and enriched rat antral mucosa gastrin cells

A technique has been developed to obtain viable, isolated and enriched populations of gastrin cells (G-cells) from the rat stomach. Restricted tissue samples from a small area of the pyloric antrum known to be particularly rich in G-cells, were sequentially digested with pronase followed by mechanical agitation, to remove the epithelial cells. This technique resulted in a significant enrichment of G-cells (3-4 fold) since the surface epithelial cells and upper portions of the glands were discarded before the initial G-cell fraction was collected. These cells in suspension were then isolated from each other by gentle pipetting in a DNase containing solution and designated the crude preparation (CP). The G-cells were then purified further by separating the cells according to size by velocity sedimentation. The greatest concentration of G-cells (15-25%) was found in the fraction containing cells with diameters of 10 to 12 micrometer. The effectiveness of the technique was evaluated by counting G-cells as identified by electron microscopy and immunofluorescence and assessing gastrin activity by radioimmunoassay. All three methods indicated that cell separation by gravity velocity sedimentation enriched the G-cell population 15-20 fold over their concentration in the CP. The combined techniques of selective pronase digestion followed by gravity velocity sedimentation resulted in an isolated cell preparation containing a 50-100 fold increase of G-cells over their normal distribution in the intact gastric mucosa. Since these isolated G-cells retain features indicating viability, their usefulness for in vitro studies is suggested.     

Vagal inhibition in the antral region of guinea pig stomach

The effects of vagal stimulation in the presence of a muscarinic antagonist were examined on three distinct rhythmically active cells located in guinea pig antrum. Vagal stimulation inhibited contractions of the circular muscle layer but did not change their rate of occurrence. With the use of intracellular recording techniques, these stimuli were found to initiate inhibitory junction potentials in the circular layer but produced smaller potential changes in driving and follower cells. Inhibition of the circular muscle layer involved two separate components. The dominant component was independent of changes in membrane potential and was abolished by nitro-L-arginine. After abolishing Ca(2+) entry into smooth muscle cells with a Ca(2+) antagonist, vagal stimulation continued to inhibit the residual contractions associated with each slow wave. When the cyclic changes in intracellular Ca(2+) concentration associated with each slow wave were measured, they were found to be unchanged by vagal stimulation. The observations suggest that vagal inhibition of stomach movements does not alter pacemaker activity in the stomach; rather, it results from a change in the sensitivity of smooth muscle contractile proteins to Ca(2+).     

Quantitation of conductance pathways in antral gastric mucosa

The magnitude of cellular and shunt conductance of Necturus gastric antral mucosa was studied by (a) comparing the cellular PD response to transepithelial PD response during changes of ionic activity in the serosal bathing solution and (b) by measurement of current spread within the epithelial sheet. Using constant product KCl changes cellular resistance was 6,788 omegacm2 and shunt resistance was 1,803 omegacm2. Deletion of HCO3- from the serosal solution produced similar but quantitatively smaller changes in PD. Using HCO3- deletion cellular resistance was 7,338 omegacm2 and shunt resistance was 1,973 omegacm2. Measurement of current spead within the mucosa avoids changing ionic gradients yet gave very similar results; cellular resistance was 8,967 omegacm2 and shunt resistance was 2,947 omegacm2. The shunt contribution to transepithelial conductance ranged from 75.2 to 79.0%. Shunt selectivity was assessed using KCl dilution potentials, where mucosal dilution gave a small change in tissue PD compatible with an anion/cation selectivity ratio of 1.16 across the shunt, whereas serosal dilution effect was dominated by a PD change across the serosal membrane of the cell.     

Dynamic histology of the antral epithelium in the mouse stomach: I. Architecture of antral units

The architecture of the pure mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice. Units were serially cut in cross section and stained by a method combining the periodic acid-Schiff sequence, a modified Grimelius's silver nitrate procedure, and Regaud's hematoxylin. A total of 195 units were then reconstructed. Of these, six were cast in polyester resin and 189 were two-dimensionally reproduced on graph paper. The reconstructions showed antral units to be divided among three main classes. The first class, which contained 32% of the units, consisted of fingerlike tubules referred to as "singlets." Three types of singlets were observed. The first or type A, which represented 76% of the singlets, was divisible into three successive portions: a pit (foveola) opening onto the mucosal surface and lined by mucous cells referred to as pit cells, an isthmus continuous with the pit and containing immature proliferative mucous cells, and a gland forming the blind end of the tubule and lined by mucous cells referred to as gland cells. Type B (14% of singlets) was similar to type A except that its gland was forked. Type C (10% of singlets) differed by the absence of a gland. The units of the second class, which contained 53% of the total number, were joined together along part of their length and were named "multiplets." Most of them (90%) were organized into clusters of two, and 10% into clusters of three. In the joined portion, the epithelial cells of the adjacent units were in contact through junctional complexes and, therefore, were not separated by basement membranes. Otherwise the units showed the same component parts as in singlets. Also, as in singlets, the majority of the units were type A and a few were type B or C. The units of the third class, or "intermediates," consisted of tubules which exhibited a branching process. This process was of variable length but could include gland, isthmus, and sometimes pit. Thus, the process duplicated a varying proportion of the unit. In conclusion, the pure mucous units of the antrum exhibit various patterns which have been designated singlet, multiplet, or intermediate. It is proposed that these three patterns are related and represent temporal differences in the duplication and production of new units. Based on this assumption, a model has been elaborated to depict the likely sequence in the proliferation of pure mucous units. It is proposed that this proliferation takes place in the antrum of young adult mice.     

Gastrin secretion by ovine antral mucosa in vitro

The effect on gastrin and somatostatin release in sheep of stimulatory and inhibitory peptides and pharmacological agents was investigated using an in vitro preparation of ovine antral mucosa. Carbachol stimulated gastrin release in a dose-dependent manner but had no effect on somatostatin release. As atropine blocked the effect of carbachol, cholinergic agonists appear to stimulate gastrin secretion directly through muscarinic receptors on the G-cell and not by inhibition of somatostatin secretion. Both vasoactive-intestinal peptide (VIP) and gastric-inhibitory peptide (GIP) increased somatostatin release but did not inhibit basal gastrin secretion, although VIP was effective in reducing the gastrin response to Gastrin-releasing peptide (GRP). Porcine and human GRP were stimulatory to gastrin secretion in high doses but bombesin was without effect. The relative insensitivity to GRP (not of ovine origin) previously reported from intact sheep may be caused either by a high basal release of somatostatin or by the ovine GRP receptor or peptide differing from those of other mammalian species.     

Dynamic histology of the antral epithelium in the mouse stomach: IV. Ultrastructure and renewal of gland cells

The renewal of gland cells was investigated by three-dimensional reconstruction of typical mucous units of the pyloric antrum using electron microscopy and 3H-thymidine radioautography in 3 to 4 month-old CD1 mice. Based on analysis of 42 units, the average gland measured 31 micron in length and was composed of 37 (mucous) gland cells with eight enteroendocrine cells scattered among them. The gland neck cells located close to the isthmus showed the cytoplasmic and nuclear features characteristic of differentiating cells. The mid-gland cells occupying the central portion of the gland appeared to be at a more advanced stage of development and completing differentiation. The gland base cells comprising the blunt end of the gland were fully mature. To quantify the renewal process, the percent of gland cell nuclei carrying label was determined at several times following 3H-thymidine administration. The rate of proliferation was found to be greatest in the gland neck, lower in the mid-gland, and even lower within the gland base. Furthermore, the isthmus contributed to gland-cell renewal by providing an estimated 12.4 cells per day. Labeled cells migrated toward the blunt end of the gland. The migration rate became progressively slower with their descent, and many cells were lost along the migration pathway, mainly in the gland neck. The loss took place without being preceded by gradual cell degeneration, but occurred as a result of rapid extrusion to the lumen or, less frequently by pyknosis, which could be followed by phagocytosis. It is concluded that the rapid rate of mitosis within the isthmus and gland neck generates a pressure causing downward migration of the cells toward the blunt end of the gland. The rate of migration, however, gradually diminishes as cells descend into the gland, presumably owing both to decreasing proliferation rate and to cell loss. Thus, while cells migrate down toward the gland base, many are lost before reaching it. This sequence is described as "the cascade pattern" of renewal.     

Dynamic histology of the antral epithelium in the mouse stomach: II. Ultrastructure and renewal of isthmal cells

The isthmus of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice using light and electron microscopy as well as 3H-thymidine radioautography. On the average, the isthmus measured 25 microns in length and was composed of 36 isthmal cells and two enteroendocrine cells. Isthmal cells generally displayed features found in embryonic cells, such as many free ribosomes, scant organelles, and a large reticulated nucleolus, and were, therefore, at an immature stage of development. Isthmal cells could be devoid of secretory granules ("granule-free cells," 2%) or contain a few small, spherical, PA-Schiff-positive, mucous granules in their apex. The granules in some of the cells had a variegated appearance and a diameter averaging 235 nm ("mottled granule cells," 39%); in other cells, the granules had a large diameter, 278 nm, with a pale background and a dense core ("core granule cells," 28%); while in still others they were homogeneously dark and measured 264 nm ("dense granule cells," 12%). Finally, some cells included a mixture of core and dense granules ("mixed granule cells," 14%). One hour after a single injection of 3H-thymidine, 37% of the isthmal cells were labeled. Each of the five isthmal cell types could acquire label and, therefore, divide. After one or more days of continuous 3H-thymidine infusion, all isthmal cells were labeled. Their turnover time was estimated to be 16.1 hr (t1/2 = 11.2 hr). The isthmus is thus composed of several cell types which are turning over rapidly. While all are relatively immature, the various types are thought to represent different developmental stages in the life history of an isthmal cell. A model devised on this basis proposes that the granule-free cells are stem cells, from which mottled granule cells are derived. These in turn evolve into either the dense granule cells of the upper isthmus or the core granule cells of the lower isthmus, or into the mixed granule cells (which are believed to develop eventually into dense granule cells or core granule cells). Maintenance of a steady state requires that the rapid production of isthmal cells be associated with rapid emigration; the dense granule cells presumably going to the pit and the core granule cells to the gland. The turnover of isthmal cells is accordingly described as following a "bidirectional pattern" of renewal.     

Dynamic histology of the antral epithelium in the mouse stomach: III. Ultrastructure and renewal of pit cells

The pit (foveola) of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice, using light and electron microscopy, sometimes combined with 3H-thymidine radioautography. Reconstruction of units from serial sections revealed that, on the average, the pit measured 151 microns in length and was lined by 184 mucus-containing pit cells. Of these, 164 were located along the wall of the pit, whereas 20 surrounded its opening on the free surface. For ultrastructural examination the pit was divided into equal thirds. The proximal third, located next to the isthmus and referred to as pit base, was composed of cells showing electron-dense mucous granules greater in number but similar in density and diameter to those of isthmal dense granule cells. Nucleoli were rather large, irregular, and reticulated; these and other features were indicative of partial differentiation. The appearance of the cells gradually changed with the distance from the isthmus. In the middle third or mid pit, cells had small, fairly rounded nucleoli, while mucous granules were more numerous than in the pit base but similar in appearance and size; these cells were considered to be mature. In the distal third or pit top-surface, granules became elongated and nucleoli shrank, and lysosomes and vacuoles greatly increased in number, indicating that cells were at a terminal stage. Indeed, some of the cells were extruded into the stomach lumen while others were phagocytosed by adjacent cells. Following a single injection of 3H-thymidine, labeling was found only in a small cohort of cells in the pit base. At the end of 1 day of continuous infusion, the cohort of labeled cells had reached the mid pit; by 2 days, the pit top; and by 3 days, the free surface, where cells were eventually lost. The renewal time of pit cells was assessed at 2.98 days (t1/2 = 1.8 days), giving a turnover rate of 33.5% per day. It is estimated that the divisions of pit base cells provide two-thirds of the cells needed daily for pit-cell renewal, while the other third is supplied by an influx of dense granule cells from the isthmus. These cells enter the pit and continuously migrate toward the gastric lumen, while differentiating in the pit base, maturing in the mid pit, and reaching a terminal stage at the pit top-surface. The progressive and orderly migration of pit cells is described as a "pipeline pattern" of renewal. It is completed in about 3 days when terminal cells are lost at the pit top-surface.     

Mast cells in the rat gastric mucosa are not primarily responsible for PGD2 generation

The present study investigates the contribution of gastric mast cells on PGD2 generation in rat gastric mucosa. Cold-restraint induced stress or i.v. carbachol injection methods were used for gastric mast cell degranulation. In 19 stressed, 15 carbachol-infused and 14 control rats, gastric mast cell counts and gastric mucosa PGD2 assay were performed. Gastric mucosal content of PGF2 alpha was also determined in carbachol infused and control rats. The mean number of gastric mast cells was significantly lower in stressed and carbachol infused than in control rats. Despite these differences in gastric mast cell counts, neither PGD2 or PGF2 alpha contents in the gastric mucosa were significantly different in mast cells degranulated rats than in control animals. These results suggest another source of PGD2 in the rat gastric mucosa other than mast cells.     

β-lipotropin-like material in human pancreas and pyloric antral mucosa

Evidence for the presence of the endogenous opioid precursor β-lipotropin has been found in human pyloric antral mucosa and pancreas by radioimmunochemical displacement curves, Sephadex chromatography, ion exchange chromatography, and immunocytochemistry. Endogenous opioids are not only present in the pituitary gland and nervous tissue but also in the stomach and pancreas, supporting the concept of a common neuro-endocrine system present in brain and gut.