Immortalization of bovine pancreatic duct epithelial cells.

Pancreatic duct cell lines have been isolated from a number of animal and human tumors, but none appear to express ion transport properties expected for differentiated pancreatic duct epithelial cells. We sought to generate an immortalized ductal cell line from well-differentiated primary cultures of bovine pancreatic duct epithelium. Epithelial cells from the main duct of the bovine pancreas were isolated and immortalized by transfection with a DNA construct encoding simian virus 40 large T antigen. A single clone (BPD1) survived negative selection and was maintained in culture for > 100 passages over 2 yr. The cells grow readily in culture as monolayers and express several properties characteristic of differentiated pancreatic ductal epithelium. The cells do not appear to form a functional tight junction complex, since the transepithelial resistance of the monolayer cultures grown on a permeable support is < 10 omega.cm2. Northern blot analysis revealed that the cells continue to express simian virus 40 large T antigen and contain significant levels of mRNA for proteins thought to be important in transepithelial bicarbonate secretion [carbonic anhydrase II, Cl-/HCO3- exchanger, Na+/H+ exchanger, and cystic fibrosis transmembrane conductance regulator (CFTR)]. In vivo pancreatic ductal secretion is stimulated by the peptide hormone secretin. The secretin receptor is expressed and functionally coupled to adenylate cyclase in the immortalized cells, since secretin caused a dose-dependent accumulation of adenosine 3'5'-cyclic monophosphate (cAMP; approximately 20-fold increase over basal levels) with a mean effective concentration of 15 nM. Elevation of intracellular cAMP by exposure of the cells to forskolin (10 microM) or secretin (0.1 microM) increase plasma membrane Cl- permeability, most likely mediated by activation of CFTR. The results of these studies demonstrate that the pancreatic duct cell line (BPD1) retains several properties exhibited by the secretory epithelial cells that line the pancreatic ductal tree. This cell line should prove useful for studies of expression, function, and regulation of pancreatic duct cell proteins.

Characterization of the mouse carbonic anhydrase II gene promoter.

This work characterizes a 250-base pair (bp) fragment of the mouse carbonic anhydrase II (CAII) gene as an efficient but not cell-specific promoter. This fragment contains multiple consensus cis regulatory regions that interact in a complex fashion to regulate transcriptional activity from the promoter. Truncated fragments of the 250-bp promoter retain transcriptional activity. The 90-bp 5' "GC"-rich portion of the promoter can direct transcription independently from the consensus TATA box and also contains a silencer that diminishes transcription from the 3' 160-bp portion of the CAII promoter as well as from the SV40 promoter. There are two nonconsensus dual functioning Ap2-like elements on the promoter that are essential for core promoter activity and cAMP-mediated increases in transcription of the gene. A nuclear protein of approximately 65 kDa binds to these elements and is present in nuclear extracts of nonstimulated and forskolin-stimulated NIH-3T3 cells. I conclude that these nonconsensus Ap2-like elements and their cognitive binding protein play a major role in the expression of the CAII gene.

Regulation of gastrin alpha-amidation in the developing rat stomach.

Because the gastrin molecule must be alpha-amidated to have maximum biological activity, rat pups from 1 to 6 wk of age were treated with dexamethasone (2 mg.kg-1.day-1) for 3 or 7 days, diethyldithiocarbamate (DDC; 400 mg.kg-1.day-1 x 3 days), dexamethasone and DDC, pentagastrin (750 micrograms.kg-1.day-1), or bombesin (40 micrograms.kg-1.day-1) for 3 days to determine the effects of these agents on alpha-amidation and gastrin and glycine extended gastrin (G-Gly) concentration in the stomach. Three day treatment with dexamethasone increased gastrin concentration by increasing amidation in pups before 5 wk of age and thereafter by enhancing preprogastrin synthesis or processing. Seven day dexamethasone treatment had no substantial effect on amidation. DDC universally inhibited amidation and affected a sustained increase in gastrin plus G-Gly concentration after the third week of life. Dexamethasone did not reverse the effects of DDC. Pentagastrin increased amidation in 1-, 3-, and 6-wk old rat pups but had no consistent effect on peptide concentration. Bombesin increased the sum of gastrin and G-Gly concentration in all but 1- and 5-wk old pups but had variable effects on alpha-amidation. We conclude that alterations in gastrin alpha-amidation have age-specific effects on tissue gastrin and G-Gly concentration and speculate that changes in tissue gastrin and G-Gly stores available for release might ultimately affect parietal cell and G-cell function during development.

Expression and post-translational processing of gastrin in heterologous endocrine cells.

The biosynthesis of gastrin involves a complex series of post-translational processing reactions that result in the formation of a biologically active secretory product. To study the mechanisms for two specific reactions in gastrin processing, namely dibasic cleavage and amidation, we infected AtT-20, GH3, and Rin5-f cells with the retroviral expression vector, pZip-NeoSV(X), containing human gastrin cDNA. We detected gastrin and its glycine extended post-translational processing intermediates (G-gly) in the media and cell extracts of successfully infected cells. Characterization of the molecular forms of gastrin in these cell lines revealed that GH3 and Rin5-f processed gastrin in a manner similar to antral G-cells but the cleavage of the Lys74-Lys75 bond that converts G34 to G17 appeared to be suppressed in AtT-20 cells. Even after conversion of this site to Arg74-Arg75 via site-directed mutagenesis, the At-20 cells synthesized G34 predominantly. All of the infected cells amidated gastrin but the gastrin/G-gly ratio, a reflection of amidation within the cells, was enhanced in GH3 and Rin5-f cells but diminished in AtT-20 cells upon treatment with dexamethasone (10(-4) M) for 3 days. The dibasic cleavage of gastrin was uneffected by dexamethasone. Our data suggest that the activities of post-translational processing reactions responsible for the synthesis of biologically active gastrin exhibit considerable tissue and substrate specificity.

H(+)-K(+)-ATPase and carbonic anhydrase II gene expression in the developing rat fundus.

H(+)-K(+)-ATPase and carbonic anhydrase II (CA II) are two enzymes that are involved in the production and secretion of the hydrogen ion by the gastric parietal cell and maintenance of intracellular pH therein. The present studies were undertaken to examine whether H(+)-K(+)-ATPase and CA II expression change in the rat fundus in association with the development of acid secretory capacity. Changes in enzyme mRNA content in the gastric fundus of developing rat pups 1-6 wk of age were evaluated using dot blots and ribonuclease protection assays. In additional studies the localization of H(+)-K(+)-ATPase and carbonic anhydrase II mRNA was examined by in situ hybridization in Formalin-fixed gastric tissues from rats 1, 3, 6, and 8 wk of age. We observed that H(+)-K(+)-ATPase mRNA content increased with age in the developing rat fundus while CA II mRNA exhibited a reciprocal decrease. These changes in enzyme mRNA were accompanied by concomitant changes in the regional distribution of the cells expressing the genes for the two enzymes. Although the changes in H(+)-K(+)-ATPase mRNA paralleled the development of acid secretory capacity, CA II mRNA levels might be regulated by the requirement for maintenance of intracellular pH during periods of cellular proliferation and by exposure of the gastric surface epithelium to the highly acidic luminal environment of the stomach.

Advanced xerophthalmia as a presenting sign in cystic fibrosis.

Xerophthalmia is a common complication of vitamin A deficiency in communities where malnutrition is found. We report on a 16-month-old infant with severe photophobia and failure to thrive. On examination, her major presenting sign was corneal xerosis, with corneal and conjunctival keratinization, and corneal stromal edema with opacification. Based on these findings, vitamin A deficiency secondary to fat malabsorption was suspected, and a workup confirmed the diagnosis of cystic fibrosis. With parenteral vitamin A supplementation, she had complete resolution of her ocular signs and symptoms. This case illustrates the value of a complete ophthalmic examination in the diagnosis of fat malabsorption syndromes.

Development of gastrin synthesis and posttranslational processing mechanisms in rats.

To examine the ontogeny of gastrin synthesis and posttranslational processing mechanisms, we utilized region-specific antisera to determine the factors that regulate the contents of gastrin and its precursors in the stomachs of developing rats. Gastrin content increased gradually from birth to a peak at 5 wk of age, after which there was a slight decrease to levels observed in mature animals. Unlike gastrin, glycine-extended precursors of gastrin (G-Gly) increased abruptly in the first day of life but thereafter increased at a steady state. Dexamethasone treatment and early weaning (at 11 days of age) rapidly increased gastrin content, whereas G-Gly content was decreased. On weaning animals to adult rat chow abruptly at an age (18 days) when the corticosteroid surge associated with the weaning process had already occurred, evidence for increases in both gastrin and G-Gly content were observed 7 days later. In contrast, administration of dexamethasone to 18-day-old rats enhanced gastrin content to a greater extent than G-Gly. These results suggest that the natural surge in corticosteroids associated with weaning may enhance gastrin amidation activity, whereas the concomitant dietary changes may exert a stimulatory effect of gastrin synthesis.

Parietal cell function of full-term and premature infants: unstimulated gastric acid and intrinsic factor secretion.

A 1-h basal collection of gastric secretions was taken from 14 healthy full-term infants (group I) and 10 premature infants (group II). Simultaneously, acid and intrinsic factor (IF) secretion was measured. In an additional group of 19 full-term infants (group III), 16 had IF detectable in their gastric secretions immediately at birth. Volumes of gastric juice (ml/kg/h) and IF secretion (ng/kg/h) were similar for group I and group II infants. Acid secretion (mEq/kg/h) was significantly less in group II (p less than 0.01). There was a linear correlation between acid secretion (mEq/kg/h) and IF secretion (ng/kg/h) for group I (r = 0.85, p less than 0.001). There was no correlation between the values for the premature infants in group II. The study documents that the parietal cell of the premature infant has the same capacity to secrete IF as the cell of the more mature infant. It also demonstrates the presence at birth of IF in gastric secretions, which suggests in utero secretion and independence from enteral feeding for stimulation.

Prostaglandin E2 concentration in gastric secretions of critically ill, full-term, and premature infants.

Critically ill, full-term, and premature infants are at increased risk to develop gastric mucosal ulceration. The cytoprotective effects of prostaglandin E2 (PGE2) may be important in the preservation of gastric mucosal integrity in these patients. PGE2 concentrations in the gastric secretions of seven full-term and 16 premature infants with severe pulmonary disease were measured by radioimmunoassay. The mean PGE2 concentration was significantly greater in the full-term infants (460 +/- 94 pg/ml), (mean +/- S.E.M.) than in the premature infants (190 +/- 35 pg/ml), P less than 0.01. There was a linear correlation between gestational age and PGE2 concentration, r = 0.70, P less than 0.001. In the 11 infants greater than or equal to 35 wk gestational age there was a linear correlation between gastric pH and PGE2 concentration, r = 0.69, P less than 0.01.

Chronic diarrhea and neutropenia not associated with pancreatic insufficiency: a non-Shwachman-Diamond entity.

Six children with chronic diarrhea and neutropenia, initially referred for evaluation of Shwachman-Diamond syndrome, were found to have no evidence of pancreatic insufficiency. All presented in the spring with a prodromal respiratory illness. Hematologic evaluation was normal except for iron deficiency anemia and neutropenia. Small intestinal biopsies of all children showed inflammation, consistent with chronic enteritis. The children were followed until growth returned to previous percentiles. Diarrhea and neutropenia resolved by 6-month follow-up, and there was no recurrence of the neutropenia at 1 year.