Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.     

Effect of omeprazole on secretion,synthesis and the gene expression of pepsinogen in the guinea pig stomach mucosa

The effects of omeprazole, a proton pump inhibitor, on gene expression, protein synthesis, intracellular storage and secretion of pepsinogen in guinea pig stomach were investigated. After treatment with omeprazole for five days, acid and pepsinogen secretion into the gastric lumen was significantly reduced. Concomitant with this, there was an increase in intracellular pepsinogen as demonstrated by increased pepsin activity in the gastric mucosa, more intense immunohistochemical staining by antibodies specific of pepsinogen and accumulation of secretory granules in the cells producing pepsinogen. In these cells, the amount for pepsinogen mRNA was reduced as revealed by Northern blotting and in situ hybridization. Ultrastructurally the endoplasmic reticulum of these cells was poorly developed, the findings being consistent with a reduction in protein synthesis. It appears that omeprazole inhibits the secretion of pepsinogen, increasing the intracellular store and leading to the reduction in gene expression probably by a feedback mechanism and consequent reduction in pepsinogen synthesis. Since these changes were most evident in the acid-secreting fundic gland mucosa, as compared with other mucosae secreting only pepsinogen, namely pyloric and duodenal mucosa, it appears probable that these changes are linked with omeprazole-induced reduction in the acid secretion.     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Identification of pepsinogen gene in the genome of stomachless fish, Takifugu rubripes

Pepsinogen is a precursor of pepsin, a gastric specific protease belonging to the aspartic proteinase family. In teleosts, several species, such as zebrafish and puffer, have independently lost gastric glands. So whether puffer have pepsinogen gene or not is an interesting issue. A search of GSS database for pufferfish, Takifugu rubripes, revealed five different aspartic proteinase genes in its genome. One of them (pPep) has typical pepsinogen structure and belongs to the fish pepsinogen cluster by phylogenic analysis. The pPep antisense probe hybridized to the gastric glands of flounder stomach. Therefore, we concluded that pPep is a pufferfish pepsinogen. The pufferfish pepsinogen mRNA was not expressed in the digestive organs but specifically in the skin. We speculated that while Tetraodontiformes evolutionarily lost gastric glands, pufferfish pepsinogen acquired an alternative function to food digestion.     

Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.     

Precocious induction of pepsinogen in the stomach of suckling mice by hormones

Effects of hormones on pepsinogen activity in mouse stomach were investigated by enzyme assay and electron microscopy. Administration of hydrocortisone alone to mice on days 5–10 increased the enzyme activity in the stomach to as much as 4.5-fold that of untreated mice and the increase was dose dependent. Thyroxine also evoked precocious differentiation of the stomach. The effects of thyroxine and hydrocortisone were additive. Injections of insulin had little effect when given alone, or in combination with other hormones. Injection of hydrocortisone alone or plus thyroxine also caused morphological differentiation of the chief cells in the stomach mucosa. Administration of thyroxine to mice on days 15–20 induced as much enzyme activity as that induced by hydrocortisone, but neither of these hormones had any effect when injected after day 23.These results suggest that besides hydrocortisone, thyroxine is also involved in differentiation of the stomach in mice for the first 20 days after birth and that the normal increase of pepsinogen activity in the stomach of mice during the late suckling period is brought about by serum glucocorticoids, possibly with thyroxine.     

Relationships between the human pepsinogen DNA and protein polymorphisms.

Pepsinogens (PGA) are the inactive precursors of pepsin, the major acid protease found in the stomach. The PGA gene family exhibits polymorphic variation in human populations that can either be demonstrated by electrophoretic analysis of the proteins or by analysis of the respective genes with cDNA probes. Here, we describe the interrelationships between the most common pepsinogen protein phenotypes and the corresponding pepsinogen haplotypes (A, B, and C) containing different combinations of the PGA3, PGA4, and PGA5 genes. We propose that this unusual genetic variation involving haplotypes that contain three, two, and one genes, respectively, is the result of molecular evolution by gene duplication.     

Cloning and expression of xP1-L,a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H⁺/K⁺-ATPase β subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.     

Increases in cellular calcium concentration stimulate pepsinogen secretion from dispersed chief cells

Intracellular calcium concentration ([Ca]i) and pepsinogen secretion from dispersed chief cells from guinea pig stomach were determined before and after stimulation with calcium ionophores. [Ca]i was measured using the fluorescent probe quin2. Basal [Ca]i was 105 +/- 4 nM. Pepsinogen secretion was measured with a new assay using 125I-albumin substrate. This assay is 1000-fold more sensitive than the widely-used spectrophotometric assay, technically easy to perform, rapid, and relatively inexpensive. The kinetics and stoichiometry of ionophore-induced changes in [Ca]i and pepsinogen secretion were similar. These data support a role for calcium as a cellular mediator of pepsinogen secretion.     

Cellular distribution of gastric chief cell protein kinase C activity: differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin.

Stimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose-dependent, 6-fold increase in pepsinogen secretion and membrane-associated PKC activity. Similarly, dose-response curves for pepsinogen secretion and the increase in membrane-associated PKC activity induced by a membrane-permeant DAG (1-oleoyl-2-acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 microM) and carbachol (0.1 microM to 1.0 mM) caused a 4-fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA- and DAG-induced pepsinogen secretion is accompanied by increased membrane-associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.     

The PAR-1-activating peptide facilitates pepsinogen secretion in rats

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.     

Interaction between the calcium and adenylate cyclase messenger systems in dispersed chief cells from guinea pig stomach. Possible cellular mechanism for potentiation of pepsinogen secretion

To determine the role of the adenylate cyclase system in potentiation of enzyme secretion, we used cholera toxin to activate adenylate cyclase before examining the effects of agents on chief cell cAMP and pepsinogen secretion. Dispersed chief cells were obtained from guinea pig stomach by fractionation of mucosal cells on a Percoll gradient. Incubation of cells with 100 nM cholera toxin for 90 min and subsequent incubation with carbachol or cholecystokinin resulted in augmentation of cellular cAMP and potentiation of pepsinogen secretion. The rate of increase in cAMP with carbachol or cholecystokinin was similar to that for the potentiated secretory response. To determine the role of changes in cell calcium on these effects, we examined the actions of the ionophore A23187. In cells preincubated with cholera toxin, A23187 augmented cAMP and caused potentiation of pepsinogen secretion. The effects of A23187, carbachol, and cholecystokinin on cells preincubated with cholera toxin were abolished by removing extracellular calcium or by adding the calmodulin inhibitor trifluoperazine. These data indicate that in chief cells preincubated with cholera toxin, secretagogue-induced increases in cell calcium concentration activate calmodulin thereby augmenting levels of cAMP and causing potentiation of pepsinogen secretion. Modulation of adenylate cyclase by changes in chief cell calcium concentration appears to be one mechanism whereby secretagogue interaction can result in potentiation of pepsinogen secretion.