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The actions of hydrochloric acid, natural PGE1, PGE2 and a synthetic commercial PGE1 preparation, Enisoprost (Searle) on pepsinogen synthesis and secretion were studied in canine chief cell monolayer cultures. Hydrochloric acid, applied directly to the apical surface of chief cells using culture plate inserts (Millipore) had no effect on secretion, nor did it affect the action of any secretagogue in the basolateral medium. All prostaglandins tested showed significant stimulation of pepsinogen secretion. Basal secretion of pepsinogen after 90 min was 9.4 (1.3)% to total initial monolayer content. At 10(-6) M, PGE1 stimulated secretion was 26.1 (3.8)%; PGE2 27.9 (4)% and Enisoprost 28.8 (4.2)% of initial pepsinogen content. Stimulations by all tested prostaglandins were additive with carbachol (10(-4) M) and CCK (10(-9) M), but not with VIP (10(-6) M), dbcAMP (10(-3) M) or forskolin (10(-6) M) responses. All three prostaglandins stimulated pepsinogen synthesis as measured by 14C labelled amino acid incorporation into pepsinogen. Time course experiments were similar to those for forskolin and showed shorter time delays between stimulus and increased synthesis rate than carbachol but longer than dbcAMP. Stimulated pepsinogen secretion was inhibited by high pepsin concentrations (greater than 800 micrograms/ml) in the medium. The inhibited abolished simultaneous carbachol induced stimulation of synthesis but prostaglandin or forskolin stimulation only after two hours. Combined with the shorter response time, as compared with carbachol, these data support our previous findings that potent stimulators of cAMP production can stimulate pepsinogen synthesis directly by stimulation of mRNA synthesis, independently from an increased secretion. The additivity of effects with carbachol or CCK and similarity with forskolin stimulated synthesis supports the suggestion that the actions of prostaglandins are mediated by cAMP. [ABSTRACT FROM PUBLISHER]

The molecular mechanisms underlying the regulation of pepsinogen A (PGA) gene expression in mammalian cells are poorly understood. In this paper we describe the structural and functional analysis of the pepsinogen A gene promoter in the pig. By genomic Southern analyses we demonstrate that, in contrast with human PGA genes which are amplified and organized in haplotypes, only a single PGA gene is present per haploid porcine genome. With the aim of identifying promoter elements mediating the gastric mucosa cell-specific transcription of the PGA gene in pig, we isolated a PGA gene from a porcine genomic library. The nucleotide sequence of the first exon and 1.7 kb of the upstream DNA region were determined and compared with the corresponding regions of the human PGA gene encoding isozymogen Pg5. In order to study the promoter activity of the PGA gene a functional assay was developed: we succeeded in obtaining primary monolayer cultures of porcine gastric mucosal chief cells, suitable for transfection. Fragments of 5'-flanking and noncoding first exon sequences of the porcine and human PGA genes were linked to the chloramphenicol acetyltransferase (CAT) gene. The transcriptional activity of these hybrid genes was assessed in transient expression assays upon transfection (lipofection) of gastric and nongastric cells. Whereas PGA 5'-flanking sequences showed no promoter activity in nongastric cell types, the DNA region from -205 to +21 was found to be sufficient to direct expression of the porcine PGA constructs in a cell-specific manner. Further deletion analysis of the proximal promoter fragment identified several regions (-205 to -167, -127 to -67 and +2 to +21) acting synergistically in the transcriptional regulation of the PGA gene. In contrast, all human PGA-CAT constructs used failed to show promoter activity in porcine gastric chief cells, indicating species-specific control of PGA gene expression. In addition, the transcriptional activity of the porcine PGA promoter in chief cells from pig was completely abolished by in vitro CpG methylation. Footprint analyses of the proximal promoter fragment using nuclear extracts from either porcine gastric mucosal chief cells or liver revealed some notable differences between both extracts, which might reflect the interaction with (a) cell-specific factor(s).

The heterogeneous group of proteinases known as pepsinogens are synthesized, stored, and upon appropriate stimulation released from gastric mucosal chief cells. Under the acidic conditions of the lumen of the stomach, the proenzymes, pepsinogens, are converted to "pepsin", which plays an important physiologic role as a digestive enzyme. The potential roles for pepsin in upper gastrointestinal diseases such as gastric or duodenal ulcer and gastroesophageal reflux disease along with the recent development of in vitro gastric gland and isolated chief cell preparations have renewed interest in the study of the control of pepsinogen synthesis and secretion. In this article the authors briefly summarize current knowledge of the biology of pepsinogens and emphasize more recent findings concerning the control of the chief cell, which is related to the synthesis and secretion of pepsinogens in vitro.

We have established monolayer cultures of human and rabbit gastric mucosal cells and of isolated rabbit gastric chief cells. These cultures were capable of de novo pepsinogen synthesis and secretion, demonstrated by electrophoresis and subsequent autoradiography of cell lysates and growth medium after culture in the presence of 14C-labelled amino acids. Cultures could be maintained for 1 week without overgrowth by fibroblasts.

We have studied the effect of tripotassium dicitrato bismuthate on the peptic activity of gastric juice, both basal and pentagastrin-stimulated, from five healthy volunteers using porcine pepsin solution as a control. Tripotassium dicitrato bismuthate showed no inhibition of the proteolytic activity of either the pure porcine or the human pepsin in the gastric juice. The ulcer healing efficacy of tripotassium dicitrato bismuthate is unlikely to be related to a gastric anti-protease effect.

In this paper the role of pepsinogen has been reviewed in its physiological and clinical aspects. Although acid secretion has traditionally received far more attention clinically and has therefore been studied in great detail, the development of cellular systems has recently seen a revival in interest of pepsinogen secretion. These systems have made it possible to study pepsinogen secretion in more detail. Although many questions remain unanswered, a picture of a stimulus-secretion coupling mechanism of the chief cell has emerged that resembles in many aspects the pancreatic acinar cell, but also possesses some unique features of its own. The chief cell monolayer culture has also made it possible to study pepsinogen synthesis, and these studies seem to have solved the old controversy of whether or not modulation of pepsinogen synthesis occurs as a result of increased secretion. It now seems that pepsinogen synthesis does indeed increase in response to stimulated secretion. In addition to physiological studies, this review has discussed clinical aspects of the human pepsinogens in various gastric disorders. The clinical implications of genetic heterogeneity of the human pepsinogens are especially intriguing. Relationships between certain PGA phenotypes and certain gastric disorders have been described and some studies have tried to evaluate the relevance of these findings for diagnostic purposes. So far, it seems that PGA phenotyping alone has only limited diagnostic value, but, in combination with serum PGA determinations, could be of additional help in the diagnosis of gastric malignancy. In addition, various studies suggest that the ratio of serum PGA and PGC levels may be helpful in determining the histological status of the gastric mucosa. A very promising possibility in solving the many problems involved in exact genotype determinations through phenotyping is the recent availability of cDNA probes. With this technique, the question of whether the association between PGA phenotypes and gastric malignancy is primary or secondary may be solved in the near future. In view of the very poor prognosis for gastric cancer, further studies concerning the relationships between gastric cancer, serum pepsinogen levels, and PGA phenotypes or genotypes will hopefully lead to the possibility of an earlier diagnosis for gastric malignancy.

The relationship between electrophoretic pepsinogen A (PGA) patterns from urine and gastric mucosa was studied in healthy volunteers and in patients with various gastric disorders. Discrepancies between urinary and gastric PGA patterns were found in 63.3% of the individuals. In 9% of the subjects with these discrepancies, the phenotype class in urine was different from that in gastric mucosa. The differences were found in all diagnostic groups. The highest frequency of differences was found in patients with gastric ulcer. The differences were not related to the serum PGA level. More than 80% of the differences were caused by a lower relative intensity of pepsinogen A fraction 5 (Pg5) in urine than in gastric mucosa. The possible origin of differences in PGA isozymogen patterns was studied by organ culture of gastric biopsies. In vitro synthesis and secretion of pepsinogens were studied by electrophoresis and autoradiography. The synthesis rate of PGA in biopsies of 1-2 mm diameter was 40-100 ng/hr. Posttranslational modification of PGA isozymogens was demonstrated. Pg2 and part of Pg4 probably are secondary products of Pg3 and Pg5, respectively. In some individuals the secretion rate of Pg3 was low compared to the other isozymogens. The conversion of Pg3 into Pg2 and the differential secretion of the isozymogens may explain some of the discrepancies between gastric and urinary PGA patterns.

Gastrotropin (GT), a protein previously isolated from porcine ileal mucosa, with a molecular mass of 14,054 daltons, was extracted from canine ileum and purified to homogeneity. The canine and porcine peptides had similar relative molecular mass, charge, hydrophobicity, and amino acid compositions. Direct Edman degradation yielded no free amino acids, indicating a blocked NH2-terminus, and a partial sequence determination of the CNBr fragments demonstrated a high degree of homology with porcine GT. Previous studies have indicated that GT is a potent enterooxyntin, and to further characterize these observations we have investigated the actions of both porcine and canine GT on isolated enriched preparations of guinea pig and dog parietal and chief cells. The results of these studies demonstrate that GT is present in more than one species and that the cellular response to porcine and canine GT is identical. The efficacies of canine and porcine GT preparations in stimulating pepsinogen secretion and [14C]aminopyrine uptake were identical and equal to those of cholecystokinin octapeptide (CCK8) and pentagastrin. GT was 100-fold more potent than either of these two major secretagogues. Maximal [14C]aminopyrine accumulation was observed with 10(-8) M GT, with an ED50 of 2 x 10(-9) M compared to pentagastrin, which caused maximal accumulation at 10(-6) M and had an ED50 of 5 x 10(-8) M. Maximal pepsinogen secretion was observed with 10(-7) M GT, with an ED50 of 10(-10) M, compared to 10(-6) M for CCK8, with an ED50 of 10(-8) M. The maximal chief cell response to GT was unaffected by the addition of CCK8 or carbachol, but responded additively with forskolin, indicating that GT uses the same transduction mechanism as CCK8 and carbachol and does not involve the activation of adenylate cyclase. The ED50 values observed with both parietal and chief cells in these studies were close to the basal circulating levels of GT (3.5 x 10(-9) M) in adult pigs. These results clearly demonstrate that GT is a potent component of the enterooxyntin factor identified in studies of the role of the small bowel in the regulation of gastric secretion.