Neural, hormonal, and paracrine regulation of gastrin and acid secretion.

Physiological stimuli from inside and outside the stomach coverage on gastric effector neurons that are the primary regulators of acid secretion. The effector neurons comprise cholinergic neurons and two types of non-cholinergic neurons: bombesin/GRP and VIP neurons. The neurons act directly on target cells or indirectly by regulating release of the hormone, gastrin, the stimulatory paracrine amine, histamine, and the inhibitory paracrine peptide, somatostatin. In the antrum, cholinergic and bombesin/GRP neurons activated by intraluminal proteins stimulate gastrin secretion directly and, in the case of cholinergic neurons, indirectly by eliminating the inhibitory influence of somatostatin (disinhibition). In turn, gastrin acts on adjacent somatostatin cells to restore the secretion of somatostatin. The dual paracrine circuit activated by antral neurons determines the magnitude of gastrin secretion. Low-level distention of the antrum activates, preferentially, VIP neurons that stimulate somatostatin secretion and thus inhibit gastrin secretion. Higher levels of distention activate predominantly cholinergic neurons that suppress antral somatostatin secretion and thus stimulate gastrin secretion. In the fundus, cholinergic neurons activated by distention or proteins stimulate acid secretion directly and indirectly by eliminating the inhibitory influence of somatostatin. The same stimuli activate bombesin/GRP and VIP neurons that stimulate somatostatin secretion and thus attenuate acid secretion. In addition, gastrin and fundic somatostatin influence acid secretion directly and indirectly by regulating histamine release. Acid in the lumen stimulates somatostatin secretion, which attenuates acid secretion in the fundus and gastrin secretion in the antrum.     

Quantitaton of gastrin and somatostatin cell populations in the antral mucosa of the rat comparative distribution and evolution through different life stages

Summary Total antral gastrin and somatostatin cell populations as well as their relative distribution pattern throughout the antrum were studied in rats with advancing age from birth time to old age. Both endocrine cell populations were estimated, after staining by immunoperoxidase technique, with a quantitative method using serial parallel strips from entire stomachs. Gastrin cells were regularly found at less than 1 h of post-natal life, but were few in number (447±82 cells). Somatostatin cells, not seen at birth, were observed in all rats at seven post-natal days; then they increased in number less rapidly but more regularly than gastrin cells. During the normal adult period, corrected gastrin cell population corresponds to about 330,000–500,000 cells and corrected somatostatin cell populations to about 130,000–200,000 cells. For the whole antrum the ratio of gastrin cell to somatostatin cell populations decreases through the rat life from 6.5 at 7 days to 1.5 in old age with a stable value, 2.5, during adult period. Examination of the topographical distribution throughout the antrum of these two populations shows that, strip per strip, their numerical ratio varies. Homogeneous values for the latter occur in the middle part of antrum and, as a rule, in each group they reflect the mean value calculated for the whole of the antrum.     

Gastrin and somatostatin in the rat antrum. The effect of removal of acid-secreting mucosa

Female rats were subjected to operations aimed at reducing the amount of oxyntic gland mucosa draining its acid secretion to the antrum. The rats were provided either with Heidenhain or Pavlov pouches reducing the oxyntic mucosa draining its secretion to the antrum by about 50% or subjected to various degrees (75, 90 and 100%) of fundectomy. Ten weeks following surgery, plasma levels of gastrin and somatostatin were assayed. At the same time, antral mucosal content of gastrin and somatostatin was determined as well as the mucosal density of these hormone-producing cells. There was a relationship between the amount of acid-secreting mucosa removed and the ensuring plasma concentration of gastrin. Thus, a stepwise increase in plasma gastrin was found with the highest levels obtained in rats subjected to 90 or 100% fundectomy. The somatostatin concentration in plasma was reduced only in rats subjected to fundectomy with the most sustained decrease in animals in which all oxyntic gland mucosa had been removed. There was also a relationship between the amount of acid-secreting mucosa removed and the gastrin content of the antral mucosa. An inverse relationship seemed to exist between antral gastrin and somatostatin concentrations. However, a significant decrease in somatostatin concentration of the antral mucosa was seen only in rats subjected to a fundectomy. The number of gastrin cells in the antral mucosa was increased in fundectomized rats only, with the largest density seen in rats deprived of all oxyntic mucosa. A corresponding decrease in the number of somatostatin cells was noticed. Our results would suggest an apparent functional relationship between antral gastrin and somatostatin cells, where the antral acid load (or pH) appears to be the major factor of physiological significance.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin. The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Summary Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

Effect of small intestinal resection on somatostatin binding to cytosol of rabbit gastric mucosa

Small bowel resection in the rabbit increased gastric (fundus and antrum) somatostatin content and decreased the number of somatostatin binding sites (but not their corresponding affinity values) in gastric (fundus and antrum) cytosol three weeks after surgery. Five weeks after resection the number of somatostatin binding sites at both fundic and antral levels as well as antral somatostatin content returned towards control values whereas the fundic concentration of the peptide remained increased. Present results together with the known inhibitory role of somatostatin on various gastric functions suggest that the gastric alterations showed by animals subjected to small bowel resection may be due, at least in part, to the observed decrease of the number of gastric somatostatin binding sites.     

A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control

Ghrelin is a 28 a.a. gastric peptide, recently identified as a natural ligand of the growth hormone secretagogue receptor (orphan receptor distinct from the receptor for growth hormone releasing hormone). In the present study, radioimmunoassay demonstrated ghrelin-like material in the rat oxyntic mucosa with moderate amounts also in antrum and duodenum. Small amounts were found in the distal intestines and pancreas. Northern blot analysis revealed abundant ghrelin mRNA in the oxyntic mucosa. Immunocytochemistry demonstrated ghrelin-immunoreactivity in endocrine-like cells in the oxyntic mucosa. Such cells occurred in low numbers also in the antrum and duodenum. The rat oxyntic mucosa is rich in endocrine (chromogranin A/pancreastatin-immunoreactive) cells, such as the histamine-rich ECL cells (65-75% of the endocrine cells), the A-like cells (20-25%) and the D cells (somatostatin cells) (10%). The ghrelin-immunoreactive (IR) cells contained pancreastatin but differed from ECL cells and D cells by being devoid of histamine-forming enzyme (ECL cell constituent) and somatostatin (D cell constituent). Hence, ghrelin seems to occur in the A-like cells. The ghrelin-IR cells in the antrum were distinct from the gastrin cells, the serotonin-containing enterochromaffin cells and the D cells. Conceivably, ghrelin cells in the antrum and distally in the intestines also belong to the A-like cell population. The concentration of ghrelin in the circulation was lowered by about 80% following the surgical removal of the acid-producing part of the stomach in line with the view that the oxyntic mucosa is the major source of ghrelin. The serum ghrelin concentration was higher in fasted rats than in fed rats; it was reduced upon re-feeding and seemed unaffected by 1-week treatment with the proton pump inhibitor omeprazole, resulting in elevated serum gastrin concentration. Infusion of gastrin-17 for 2 days failed to raise the serum ghrelin concentration. Omeprazole treatment for 10 weeks raised the level of HDC mRNA but not that of ghrelin mRNA or somatostatin mRNA in the oxyntic mucosa. Hence, unlike the ECL cells, ghrelin-containing A-like cells do not seem to operate under gastrin control.     

Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract

 Colocalisation of synaptophysin has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum and colon) using double-immunofluorescence stainings. Numerous synaptophysin immunoreactive cells were seen in the antrum, while a smaller number were found in the intestinal tract. Synaptophysin immunoreactivity was strong in the antrum but weak in the intestine. In the intestinal colocalisation studies the synaptophysin immunoreactivity was enhanced by using the tyramide amplification method. Synaptophysin and chromogranin A were colocalised but the latter occurred mainly basally, whereas synaptophysin was found to occur diffusely throughout the cytoplasm. Synaptophysin immunoreactivity occurred in the serotonin cells throughout the gastrointestinal tract, and in the antral gastrin and somatostatin cells. In the intestinal tract only a small fraction of somatostatin, gastrin, cholecystokinin, enteroglucagon, enteroglucagon/ peptide tyrosine tyrosine displayed synaptophysin immunoreactivity. In the gastrointestinal tract (except the antrum), chromogranin A is a better general neuroendocrine marker than synaptophysin. The functional role of synaptophysin is unclear but it may be involved in the intracellular transport and release of hormones. Based on the distribution background of synaptophysin, it seems to be of greater importance in the antrum than in the intestinal tract as a whole. Accepted: 3 September 1998     

Somatostatin stimulates menin gene expression by inhibiting protein kinase A

Somatostatin is a potent inhibitor of gastrin secretion and gene expression. Menin is a 67-kDa protein product of the multiple endocrine neoplasia type 1 (MEN1) gene that when mutated leads to duodenal gastrinomas, a tumor that overproduces the hormone gastrin. These observations suggest that menin might normally inhibit gastrin gene expression in its role as a tumor suppressor. Since somatostatin and ostensibly menin are both inhibitors of gastrin, we hypothesized that somatostatin signaling directly induces menin. Menin protein expression was significantly lower in somatostatin-null mice, which are hypergastrinemic. We found by immunohistochemistry that somatostatin receptor-positive cells (SSTR2A) express menin. Mice were treated with the somatostatin analog octreotide to determine whether activation of somatostatin signaling induced menin. We found that octreotide increased the number of menin-expressing cells, menin mRNA, and menin protein expression. Moreover, the induction by octreotide was greater in the duodenum than in the antrum. The increase in menin observed in vivo was recapitulated by treating AGS and STC cell lines with octreotide, demonstrating that the regulation was direct. The induction required suppression of protein kinase A (PKA) since forskolin treatment suppressed menin protein levels and octreotide inhibited PKA enzyme activity. Small-interfering RNA-mediated suppression of PKA levels raised basal levels of menin protein and prevented further induction by octreotide. Using AGS cells, we also showed for the first time that menin directly inhibits endogenous gastrin gene expression. In conclusion, somatostatin receptor activation induces menin expression by suppressing PKA activation.     

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.     

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.     

The effects of gastrin, epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6)

Exposure of IEC-6 cells for 24 hr to either gastrin (50-500 ng/ml) or EGF (100-500 ng/ml) significantly stimulated (100-165%) the rate of [3H]thymidine incorporation into DNA (referred to as DNA synthesis) when compared with the corresponding basal levels. Somatostatin (10-500 ng/ml) produced no apparent change in DNA synthesis in IEC cells. On the other hand, somatostatin completely inhibited the EGF-induced rise in DNA synthesis. The gastrin-mediated stimulation in DNA synthesis was not affected by somatostatin. The rate of DNA synthesis in IEC cells in the presence of both gastrin and EGF was found to be greater (additive) than that caused by either of the peptides alone. A similar but less dramatic change in the actual number of cells (assessment of cell replication) was observed when the IEC cells were exposed for 24 hr to gastrin, EGF, and somatostatin, either alone or in combination. Whereas gastrin (250 ng/ml) and EGF (250 ng/ml) by themselves increased the number of cells significantly by 29 and 37%, respectively, together they caused a 72% stimulation, when compared with the basal levels. Somatostatin by itself caused no apparent change in IEC cell population, but it significantly inhibited the EGF- but not the gastrin-induced stimulation in IEC cell replication. It is concluded that both gastrin and EGF exert a direct proliferative effect on IEC cells, and the EGF action is regulated by somatostatin.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33-49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense "halo" of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophys. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Ultrastructure of the gut neurotensin cell

Summary In mammals, neurotensin cells occur scattered in the epithelium of the jejunum-ileum. In chicken, neurotensin cells are abundant in the region of the gizzard-duodenal junction (antrum) where they occur intermingled with numerous somatostatin and gastrin cells. The neurotensin cells in chicken, dog and man were identified at the electron microscopic level by immunocytochemistry, using the consecutive semithin/ultrathin section technique. They contain numerous electron dense cytoplasmic granules, predominantly in the basal portion of the cell. It was shown that these granules are the storage site for neurotensin. The neurotensin granules are round, highly electron dense and of about the same size in the different species examined (mean diameter 260–290 nm). in dog and man the granules have a tightly applied surrounding membrane while in the chicken a relatively electron lucent zone separates the electron dense core from the granule membrane. The ultrastructure of the neurotensin granules in chicken is some-what reminiscent of that of the gastrin granules. The mean diameter of the gastrin granules in chicken antrum is 230 nm; for the somatostatin granules the mean diameter is 305 nm.     

Ultrastructure of the gut neurotensin cell.

In mammals, neurotensin cells occur scattered in the epithelium of the jejunum-ileum. In chicken, neurotensin cells are abundant in the region of the gizzard-duodenal junction (antrum) where they occur intermingled with numerous somatostatin and gastrin cells. The neurotensin cells in chicken, dog and man were identified at the electron microscopic level by immunocytochemistry, using the consecutive semithin/ultrathin section technique. They contain numerous electron dense cytoplasmic granules, pre-dominantly in the basal portion of the cell. It was shown that these granules are the storage site for neurotensin. The neurotensin granules are round, highly electron dense and of about the same size in the different species examined (mean diameter 260--290 nm). In dog and man the granules have a tightly applied surrounding membrane while in the chicken a relatively electron lucent zone separates the electron dense core from the granule membrane. The ultrastructure of the neurotensin granules in chicken is somewhat reminiscent of that of the gastrin granules. The mean diameter of the gastrin granules in chicken antrum is 230 nm; for the somatostatin granules the mean diameter is 305 nm.     

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.