Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse

Summary The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was carried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simultaneous immunolabelling of various gastro-pancreatic hormones for topographical comparison. In the rat stomach, nerve fibers displaying intense GAD-like immunoreactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submucous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corresponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD-like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stomach but that it could also be an endocrine or paracrine factor in the stomach and pancreas.     

Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse.

The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was carried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simultaneous immunolabelling of various gastro-pancreatic hormones for topographical comparison. In the rat stomach, nerve fibers displaying intense GAD-like immunoreactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submucous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corresponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD-like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stomach but that it could also be an endocrine or paracrine factor in the stomach and pancreas.     

Apoptosis in rat gastric antrum: evidence that regulation by food intake depends on nitric oxide synthase.

The turnover of the epithelium of the gastrointestinal tract is regulated by a balance between cell multiplication and cell loss. We examined the effects of starvation on apoptosis in endocrine and other epithelial cells of rat antropyloric mucosa. Apoptosis was determined by the TUNEL reaction combined with immunocytochemical staining for gastrin and somatostatin. Apoptotic cell morphology was determined by bisbenzimide staining for DNA. Both gastrin and somatostatin cells showed a significantly lower apoptotic index than the general epithelium. This agrees with the longer turnover kinetics of gastric endocrine cells. On starvation, the apoptotic index of the general epithelium and of the gastrin but not of the somatostatin, cells increased significantly. This was prevented by the nitric oxide synthase (NOS) inhibitor L-NAME but not by its inactive stereoisomer D-NAME. Immunoreactive neuronal NOS was present in somatostatin cells, in nonendocrine cells predominating in the surface and pit epithelium, and in rare nerve fibers. Endothelial cell NOS was present in vessels, whereas the inducible isoform was barely detectable. Thus, endogenous NOS isoforms participate in regulating antropyloric epithelial apoptosis during starvation. The close paracrine relation between somatostatin cells and gastrin cells suggests that the former regulates apoptosis of the latter through release of NO.     

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach.

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.     

Coexpression of the gastrin and somatostatin genes in differentiating and neoplastic human cells

Double immunofluorescence and in situ hybridizations performed on adjacent thin sections show that a population of normal antropyloric cells of the human stomach expresses both gastrin and somatostatin mRNA's and the corresponding peptides. Such cells were present in both adult and fetal antropyloric mucosa and were situated in the regenerative (isthmus) region of the antropyloric tubes. It is, hence, likely that these cells represent immature endocrine cells that yet have to be committed to either the gastrin or somatostatin lineage. Cells coexpressing gastrin and somatostatin were also detected in pancreatic endocrine tumours. The presence of gastrin-somatostatin cells during development and in tumours suggests that gastrin and somatostatin cells may differentiate from such multipotent precursor cells.Presented in part at the 76th Annual Meeting of the Endocrine Society, 15–18 June 1994, Anaheim, Calif., USA, Abstract no. 691     

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.     

Pancreatic-duodenal homeobox 1 -role in gastric endocrine patterning

The gastrointestinal tract is subdivided into regions with different roles in digestion and absorption. How this patterning is established is unknown. We now report that the pancreatic-duodenal homeobox 1 gene (pdx1) is also expressed in cells of the distal stomach. Positive cells include subpopulations of the three main endocrine (gastrin, somatostatin and serotonin) cell types of this region. Pdx1 deficient mice were virtually devoid of gastrin cells, had normal numbers of somatostatin cells and increased numbers of serotonin cells. Pdx1 is thus important for development of the gastrin cells of the antropyloric mucosa of the stomach and probably acts by controlling the fate of gastrin/serotonin precursor cells.     

Topography of somatostatin cells in the stomach of the rat: Possible functional significance

Summary Somatostatin cells in the stomach of the rat have a characteristic shape and distribution. In the antral mucosa they occur together with gastrin cells and enterochromaffin cells at the base of the glands. In the oxyntic mucosa they are scattered along the entire glands with some predominance in the zone of parietal cells. Throughout the gastric mucosa the somatostatin cells possess long and slender processes that emerge from the base of the cell and end in clublike swellings. Such processes appear to contact a certain proportion of neighbouring gastrin cells in the antral mucosa and parietal cells in the oxyntic mucosa.Exogenous somatostatin given by intravenous infusion to conscious rats counteracted the release of gastrin stimulated by feeding, elevated antral pH or vagal excitation. Gastrin causes parietal cells to secrete HCl and endocrine cells in the oxyntic mucosa to mobilise and synthesise histamine. Somatostatin is known to block the response of the parietal cells to gastrin. In contrast, somatostatin did not block the response of the histamine-storing endocrine cells to gastrin, perhaps because these endocrine cells lack receptors to somatostatin. Conceivably, somatostatin in the gastric mucosa has a paracrine mode of action. The observations of the present study suggest that somatostatin may affect some, but not all of the various cell types in the stomach. Under physiological conditions this selectivity may be achieved in the following ways: 1) Communication may be based on direct cell-to-cell contact. 2) Only certain cell types are supplied with somatostatin receptors.     

The components required for amino acid neurotransmitter signaling are present in adipose tissues

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABA(A)Ralpha2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues.     

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.     

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.     

Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach

There are now increasing evidences suggesting that GABA is able of direct interaction with certain endocrine cells. In the present study, highly specific anti-GABA-glutaraldehyde antibodies and 3H-GABA uptake were used at the light and electron microscope levels to investigate the occurrence of cells containing endogenous GABA or taking up exogenous GABA in the mucosal antrum and corpus of the rat stomach. Only certain endocrine cell types of both regions were immunostained or grain-labelled. However, the morphology of their secretory granules did not allow to identify the nature of their hormone with certainty but suggested that somatostatin-like cells could interact with GABA. The combination of gastrin and somatostatin immunodetection with 3H-GABA uptake autoradiography at the light microscope level, revealed that a subpopulation of somatostatin-like cells and other still unidentified endocrine cells are able to take up GABA, while the gastrin-like cells are not. These results reinforce the hypothesis that certain endocrine cell types of the diffuse endocrine system of the digestive tract are able to directly interact with GABA.     

Regulation of histamine release from oxyntic mucosa.

The regulation of histamine release from oxyntic mucosa is complex because of two potential sources of histamine: mast cells and enterochromaffin-like (ECL) cells. A gastrin-responsive histamine pool was identified in the rat oxyntic mucosa two decades ago, but these ECL cells from the rat have not yet been isolated or characterized in vitro. In vivo studies in canine and human mucosa have been more difficult because of the high content of histamine in mast cells. Using enzyme-dispersed canine oxyntic mucosal cells, we have studied regulation of histamine release from a mast cell-depleted fraction prepared by sequential elutriation and density gradient. Histamine-like immunoreactivity was demonstrated, using peroxidase-anti-peroxidase immunohistochemistry. After short-term culture, histamine was released in response to gastrin, cholecystokinin, carbachol, and forskolin. Somatostatin potently and effectively inhibited the response to gastrin. The cultures used for these studies also contained somatostatin cells, and, furthermore, the response to gastrin was enhanced by incubation with monoclonal antibodies to somatostatin. The latter findings suggested that somatostatin was acting in these cultures by a paracrine route. This pattern contrasts with that obtained in previous studies of canine oxyntic mucosal mast cells.     

Control of rat gastric somatostatin release by gamma-aminobutyric acid (GABA)

The influence of gamma-aminobutyric acid (GABA) on gastric somatostatin and gastrin release was studied using an isolated perfused rat stomach preparation. GABA dose-dependently inhibited somatostatin release (maximal inhibition of 44% at 10(-5)M GABA), whereas gastrin secretion was not affected. The GABA agonist muscimol led to a decrease in somatostatin release of similar magnitude. The GABA-induced changes were partially reversed by 10(-5)M atropine. Gastrin secretion was not influenced by either protocol. It is concluded that GABA as a putative neurotransmitter in the enteric nervous system is inhibitory to rat gastric somatostatin release in vitro via cholinergic pathways.     

Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach

Summary There are now increasing evidences suggesting that GABA is able of direct interaction with certain endocrine cells. In the present study, highly specific anti-GABA-glutaraldehyde antibodies and ³H-GABA uptake were used at the light and electron microscope levels to investigate the occurrence of cells containing endogenous GABA or taking up exogenous GABA in the mucosal antrum and corpus of the rat stomach. Only certain endocrine cell types of both regions were immunostained or grain-labelled. However, the morphology of their secretory granules did not allow to identify the nature of their hormone with certainty but suggested that somatostatin-like cells could interact with GABA. The combination of gastrin and somatostatin immunodetection with ³H-GABA uptake autoradiography at the light microscope level, revealed that a subpopulation of somatostatin-like cells and other still unidentified endocrine cells are able to take up GABA, while the gastrin-like cells are not. These results reinforce the hypothesis that certain endocrine cell types of the diffuse endocrine system of the digestive tract are able to directly interact with GABA.     

Histamine and histidine decarboxylase are hallmark features of ECL cells but not G cells in rat stomach

The oxyntic mucosa of the rat stomach is rich in ECL cells which produce and secrete histamine in response to gastrin. Histamine and the histamine-forming enzyme histidine decarboxylase (HDC) have been claimed to occur also in the gastrin-secreting G cells in the antrum. In the present study, we used a panel of five HDC antisera and one histamine antiserum to investigate whether histamine and HDC are exclusive to the ECL cells. By immunocytochemistry, we could show that the ECL cells were stained with the histamine antiserum and all five HDC antisera. The G cells, however, were not stained with the histamine antiserum, but with three of the five HDC antisera. Thus, histamine and HDC coexist in the ECL cells (oxyntic mucosa) but not in G cells (antral mucosa). Western blot analysis revealed a typical pattern of HDC-immunoreactive bands (74, 63 and 54 kDa) in oxyntic mucosa extracts with all five antisera. In antral extracts, immunoreactive bands were detected with three of the five HDC antisera (same as above); the pattern of immunoreactivity differed from that in oxyntic mucosa. Food intake of fasted rats or treatment with the proton pump inhibitor omeprazole raised the HDC activity and the HDC protein content of the oxyntic mucosa but not of the antral mucosa; the HDC activity in the antrum was barely detectable. We suggest that the HDC-like immunoreactivity in the antrum represents a cross-reaction with non-HDC proteins and conclude that histamine and HDC are hallmark features of ECL cells but not of G cells.     

Pentagastrin and adenosine 3',5'-cyclic monophosphate stimulated secretion of somatostatin from perfused rat gastric mucosa.

Immunoreactive somatostatin is secreted by rat gastric mucosa perifused $\text{in vitro}$. Somatostatin release is stimulated by pentagastrin and cyclic AMP with theophylline. These results suggest that gastric mucosal somatostatin may have a paracrine action as feedback inhibitor of gastrin secretion.