Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures.

Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.

Culture of rat renal medullary tissue in media made hyperosmotic with NaCl and urea.

During antidiuresis, the rat kidney maintains a variable and steep osmotic gradient from the cortex (300 mOsm) to the inner medulla (at least 2,600 mOsm). Therefore, cells in the renal medulla must be able to adapt to a variably hyperosmotic environment. We have examined the ability of tissue fragments taken from various points on the cortical-medullary axis to survive and grow when cultured in media made hyperosmotic with urea and NaCl. Survival and growth were measured by the explants' ability to produce epithelial outgrowths. At osmotic concentrations of 1,100 and 1,200 mOsm, only explants from the inner medulla produced epithelial outgrowths. At 700 mOsm, all explants produced outgrowths but outgrowth size was a function of position on the cortical-medullary axis, with inner medullary fragments producing the largest outgrowths. Growth was most rapid at all osmolalities when the Na+:urea ratio was 1:1. These results are consistent with the hypothesis that renal medullary cells are adapted to elevated concentrations of Na+ and urea. Both explants and epithelial outgrowths were examined using light and electron microscopy. Physical continuities between the epithelial outgrowths and collecting duct epithelium in the explants, as well as the ultrastructural characteristics of the outgrowths at 700 mOsm, indicated that the outgrowths may have originated from collecting duct epithelium.

Isolation and culture of rhesus monkey pancreatic ductules and ductule-like epithelium.

The objective of this work was to devise methods for the isolation and culture of duct epithelium from rhesus monkey pancreas with the expectation that such methods would be applicable to the human pancreas. This objective is important because of the role duct epithelium appears to play in human diseases such as pancreatic cancer and cystic fibrosis. Pieces of freshly procured pancreas were minced and enzymatically dissociated, resulting in a digest that contained a few isolated ductules (intralobular ducts) as well as numerous small tissue fragments consisting of roughly equal proportions of ductular and acinar cells. These fragments were suspended in a rat tail collagen gel and cultured for up to 2 weeks in a medium supplemented with cholera toxin, epidermal growth factor, and other additives. A few cystic ductular fragments were initially observed among a large number of predominantly solid fragments. Later, most of the solid fragments also became cystic and eventually resembled the ductules except for being spherical. Autoradiographic analysis of DNA synthesis showed that the cysts possessed a proliferative potential. The cysts consisted almost entirely of ductule-like epithelium with no recognizable acinar cells, and exhibited greatly reduced concentrations of the acinar marker enzymes amylase, chymotrypsin, and gamma-glutamyl transferase. In contrast, the specific activity of the duct marker enzyme carbonic anhydrase was elevated in freshly isolated digests compared with the whole pancreas and this elevated activity was maintained for 4-5 days of culture, after which it declined. Other evidence for the ductular nature of the cysts was their low density relative to freshly isolated acinar tissue, their ability to distend (suggestive of fluid/electrolyte secretion), and the accumulation of mucins at the apical borders of the cells. The results show that fragments of rhesus monkey pancreas that are enriched in ductular epithelium assume some of the properties of ductular cells when cultured in a collagen gel. These epithelial preparations should facilitate biochemical and physiological studies of this important pancreatic cell type.

Histochemical distribution of carbonic anhydrase in rat and rabbit lacrimal gland.

PURPOSE: The purpose of this study was to examine the histochemical distribution of carbonic anhydrase (CA) in lacrimal glands from rats and rabbits; and to determine if age- and/or sex-related differences exist in the amount and distribution of CA in the rat lacrimal gland. METHODS: Lacrimal glands from young (3-12 wk) and aged (2-2.5 yr), male and female F344 rats and male rabbits were fixed in 1% paraformaldehyde and embedded in glycolmethacrylate. CA histochemistry was performed on 2-microns sections. The distribution of CA activity was determined by morphometric analysis. RESULTS: In rat lacrimal gland, CA activity was distributed in a discontinuous, mosaic fashion among the acinar cells. In tissue from young males and females as well as from aged females, about 10% of the acinar tissue displayed CA activity. Significantly more activity was present in tissue from aged male rats. CA was present in the ductal lumina, suggesting that it is a secretory product of the acinar cells. In rabbits, CA activity was associated with the basolateral membranes of the terminal acinar cells only. CONCLUSIONS: In rat, the presence of CA activity in certain acinar cells and in ductal lumina suggests that CA is actively secreted by the lacrimal gland. An age-related increase in the amount of CA activity in the male glands exists that may be under gender-specific hormonal influences. In the rabbit lacrimal gland, the membrane-associated CA found uniquely with the terminal acinar cells suggests that these cells have special transport functions associated with the primary secretion of lacrimal fluid.

The pancreatic duct cell.

A conference entitled "The Pancreatic Duct Cell: Physiology and Pathophysiology" was held September 26-29, 1991, at the Engineering Society Club of Baltimore. The conference was organized by a committee consisting of John Williams of the University of Michigan (Co-Chair), Daniel Longnecker of Dartmouth Medical School (Co-Chair), Barry Agent of Newcastle Upon Tyne, Raymond Frizzell of the University of Alabama at Birmingham, Sherwood Githens of the University of New Orleans, and Sarah Kalser of the NIDDK. The meeting was sponsored by the NIDDK with contributions from NCI, NIDR, ADAMHA, and the American Gastroenterological Association. About 100 investigators from the United States, England, Canada, Germany, Norway, and Israel attended the conference. The participants were based in a number of distinct disciplines including both basic and clinical sciences. While the main focus was on pancreatic ducts, comparison of salivary and bile ducts was also included.

Carbonic anhydrase II gene expression in mouse pancreatic duct cells.

Our goal is to create a transgenic mouse model for human pancreatic duct cell adenocarcinoma using the promoter/enhancer region of the carbonic anhydrase (CA) II gene to drive the expression of SV-40 T-antigen in pancreatic duct cells. This requires that the CA II gene be expressed in mouse pancreatic duct cells and not in other pancreatic cells, as has already been shown to be the case in the human and guinea pig pancreas. We have shown with an enzyme histochemical assay that mouse pancreatic duct cells contain CA activity in both intact pancreas and cultured interlobular duct epithelium. In addition, CA activity was detected with a biochemical assay in homogenates of cultured duct epithelium. The specific activity of duct cells was 2.75-fold greater than in whole pancreas, suggesting that a substantial amount of total pancreatic CA activity is contributed by duct cells. At least some of the CA in cultured duct cells was inferred to be CA II by Northern blot analysis of RNA extracted from the cells. The concentration of CA II mRNA in the cultured duct cells was substantially greater than in whole pancreas and would appear to account for the majority, if not all, of the CA II in the mouse pancreas.

Rat pancreatic duct epithelium cultured on a porous support coated with extracellular matrix.

A method was developed for the isolation and culture of rat pancreatic duct epithelium of predominantly interlobular duct origin. Purified duct epithelial fragments were cultured on a porous support (HATF filters, Millipore) at 37 degrees C in a 1:1 mixture of Dulbecco's Modified Eagle's and Ham's F-12 media supplemented with insulin, cholera toxin, epidermal growth factor, bovine pituitary extract (BPE), and Nu-Serum (Collaborative Research) in a humidified atmosphere of 95% air and 5% CO2. The filters were coated with an extracellular matrix of either rat tail collagen or Matrigel (Collaborative Research), both of which significantly enhanced growth of the duct epithelium in comparison with untreated filters. The cells grew from the tissue fragments as epithelial islands, which merged to form a confluent sheet of epithelium covering at least 80% of the filter within 10 days in culture. The mitotic index of the spreading epithelium increased with time, reaching a maximum of 0.6% on days 3 and 5 and then declining. The epithelial monolayer consisted of tightly packed cells, with a few large cells and a few cells undergoing abnormal mitoses. Fibroblast contamination was negligible. The cells retained carbonic anhydrase activity, consistent with their pancreatic ductal origin and with the maintenance of differentiation in culture. The epithelium could be subcultured but with a low efficiency. A defined, serum-free medium was established with the addition of ethanolamine, bovine serum albumin, and transferrin and the deletion of serum and BPE. The epithelial cells grew nearly as well in this medium as in the serum-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The developmental accumulation of gamma-glutamyl transferase in the pancreas of the rat.

The developmental accumulation of pancreatic exocrine secretory enzymes is well defined, but little is known of the development of other enzymes in the pancreas. This report focuses on the developmental accumulation of gamma-glutamyl transferase (GGT), a membrane-bound ectoenzyme whose specific activity in the pancreas is the second largest among rat organs. GGT activity is large in organs with active glutathione metabolism. Pancreatic GGT specific activity increased 100-fold from prenatal day 14 to birth, decreased 3-fold until about postnatal week 2, and then increased until the adult value was reached 4 weeks after birth. There was a 500-fold increase in specific activity from prenatal day 14 to the adult. The developmental accumulation pattern of GGT was very similar to that of the exocrine secretory enzyme amylase, which increased 1,300-fold from prenatal day 14 to birth, decreased 8-fold by postnatal week 1, and then increased to the adult level soon after week 4. The overall increase in amylase specific activity was 1,100-fold. The similar developmental accumulation patterns of GGT and amylase suggested that their accumulation might be regulated in a similar fashion. Although the thymidine analogue 5-bromodeoxyuridine inhibited the prenatal accumulation of amylase, as previously reported, it did not inhibit prenatal GGT accumulation. Therefore, the prenatal accumulation of GGT appears to be regulated differently than amylase. On the other hand, the postnatal levels of GGT appear to be controlled by glucocorticoids in a fashion similar to the previously reported control of amylase levels, since both enzymes could be induced to rise prematurely to adult levels by a series of three injections of the glucocorticoid dexamethasone beginning on days 7, 8, 9, 10, 11, or 12.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione metabolism in the pancreas compared with that in the liver, kidney, and small intestine.

The pancreas plays a major role, along with the kidney, liver, small intestine, and several other organs, in glutathione (GSH) metabolism, as evidenced by the large concentration of GSH in the pancreas, its rapid turnover rate, and the presence, at significant levels, of various enzymes involved in GSH metabolism. The pancreas appears to obtain much of the cysteine that is required for both GSH and protein synthesis by hydrolyzing plasma GSH to its constituent amino acids and then transporting cysteine into the cells. GSH hydrolysis is accomplished by the ectoenzymes gamma-glutamyl transferase (GGTase) and aminopeptidase N, both of which are present in the pancreas. Only the kidney has a greater GGTase activity. Although pancreatic GSH synthesis has not been directly demonstrated, pancreatic secretory protein synthesis is substantial, and these proteins contain significant amounts of cysteine as disulfides. The pancreas also contains significant levels of protein disulfide isomerase, glutathione peroxidase, and NADPH:GSH oxidoreductase. Protein disulfide isomerase, using oxidized glutathione generated by glutathione peroxidase, is important in the formation of disulfide bonds in secretory proteins in the pancreas. No other organ has a higher specific activity of protein disulfide isomerase. By analogy with kidney and liver, the pancreas presumably exhibits a rapid apical secretion of GSH. The purpose of this apical secretion is unknown in the kidney. In the liver, it is important in bile secretion. The large GGTase activity of apical plasma membranes in the pancreas is likely to be instrumental in the hydrolysis, and subsequent recovery of the constituent amino acids of apically secreted GSH, as occurs in the kidney and liver.

Rat renal papillary tissue explants survive and produce epithelial monolayers in culture media made hyperosmotic with sodium chloride and urea.

The capacity of papillary cells to adapt to elevated osmotic concentrations is unusual among mammalian cells. This capacity was evaluated by using primary tissue culture. Viability and growth of cells in rat renal papillary tissue explants were assessed after culture in media adjusted with urea and sodium chloride to various osmotic concentrations between 300 and 1,500 mOsm/kg water. The survival of cells, including cells resembling those of the collecting ducts and the loop of Henle, was greatest in medium adjusted to 1,000 mOsm with equiosmolar amounts of the two solutes. At 1,500 mOsm only cuboidal tubular epithelium resembling collecting duct epithelial cells survived. In contrast, cells of cortical tissue survived and grew at 300 and 640 mOsm, but not at 1,000 mOsm or above. Epithelial monolayers appeared to proliferate from collecting ducts and spread over the surface of the explants as well as onto the glass surface in the culture dish. Epithelial growth of medullary tissue was most rapid at 300 mOsm and was slower at 700 and 1,000 mOsm. Monolayers did not form at 1,500 mOsm; however, epithelial overgrowth of explants did occur. Hydropenia in the donor animal did not significantly affect the viability or growth of cultured papillary tissue. Explants cultured for 5 days at 300 mOsm followed by a stepwise increase in medium osmolality to 1,100 or 1,500 mOsm and cultured for 3 more days showed low or no survival whereas explants cultured at 700 mOsm survived such increases. Explants cultured for 5 days at 1,500 mOsm survived and grew monolayers when lowered to 300 mOsm. Poor viability and no epithelial proliferation were observed in explants cultured in medium adjusted to 900 mOsm with either urea or sodium chloride alone, suggesting that a mixture of the two solutes in the extracellular space, as found in vivo, may be essential in achieving elevated osmolalities.

How different DNA sequences are recognized by a DNA-binding protein: effects of partial proteolysis.

MDBP is a sequence-specific DNA-binding protein from mammals that recognizes a variety of DNA sequences, all of which show much homology to a partially palindromic 14 base-pair consensus sequence. MDBP subjected to limited proteolysis and then incubated with various specific oligonucleotide duplexes yielded two types of complexes. The relative concentrations of these complexes varied greatly depending on how closely the MDBP site matched the consensus sequence. No such DNA sequence-specific differences in the types of complexes formed were seen with intact MDBP. Partial proteolysis also changed the relative affinity of MDBP for several of its binding sites. The nature of the two types of complexes formed from fragmented MDBP and DNA was studied by DNA competition assays, protein titration, site-directed mutagenesis, and dimethyl sulfate and missing base interference assays. The results suggest that, for some specific DNA sequences, half-site interactions with one MDBP subunit predominate and for others, strong interaction of two subunits with both half-sites readily occur.

Rat pancreatic interlobular duct epithelium: isolation and culture in collagen gel.

Interlobular duct fragments from the pancreas of the rat were isolated by collagenase digestion and filtration, embedded in a matrix of rat-tail collagen, and cultured in a 1:1 mixture of Dulbecco's minimal essential and Ham's F12 media supplemented with cholera toxin (CT, 100 ng/ml) and epidermal growth factor (EGF, 10 ng/ml) in addition to supplements used previously, thereby improving the yield of ducts by a factor of two compared with previous results. The ducts were harvested by digestion of the collagen matrix with collagenase and were then dissociated by treatment with EDTA in divalent cation-free salt suspended in collagen and cultured as were the ducts. Numerous cysts appeared as a function of time and some of these enlarged dramatically. Some of the larger cysts exhibited secondary tubular processes extending into the surrounding collagen. The addition of bovine pituitary extract (BPE, 50 micrograms/ml) doubled the number of cysts, whereas omission of serum or CT + EGF reduced the number. BPE or forskolin could substitute effectively for CT. Agents that stimulate (secretin) or inhibit (e.g., ouabain or acetazolamide) fluid-electrolyte secretion in vivo had no effect on the number or average diameter of the cysts. The cysts were 83 to 88% epithelial with the balance of the cells being fibroblastic in appearance. Some cysts consisted only of epithelium. The proliferative capacity of the cystic epithelium was shown by the presence of mitotic figures and by an autoradiographic labeling index of 22 to 30% after a 24-h exposure to [3H]thymidine. The labeling index was reduced by the omission of CT + EGF. Transmission electron microscopy showed that the cysts exhibited morphologic features of duct epithelium in vivo, including apical microvilli, lateral interdigitations of the plasma membrane, and typical cytoplasmic organelles.

The pancreatic duct cell: proliferative capabilities, specific characteristics, metaplasia, isolation, and culture.

The pancreatic duct cell, although a minor cell type of the pancreas, plays an important role in fluid/electrolyte and mucin secretion, and has been implicated in the development of pancreatic cancer, alcoholic pancreatitis, and cystic fibrosis. In the normal pancreas, the duct cell has the same low proliferative rate as acinar and endocrine cells. Under certain pathological circumstances, duct cells, as well as acinar and islet cells, may be stimulated to proliferate more rapidly. Pancreatic duct cells exhibit certain features not shared by acinar and/or endocrine cells, including a variety of antigens, mucins, enzymes, and morphological features. Adult duct cells resemble fetal pancreatic duct-like cells morphologically, but they have differentiated to at least a limited extent from their precursor cell type. Although there is no evidence that duct cells differentiate into acinar cells after pancreatic morphogenesis is complete, some islet cells develop from duct epithelium in the early postnatal period. Some pathological conditions may lead to the postnatal formation of islet cells from duct cells and may cause acinar cells to become duct-like in morphology or to die and be replaced by duct cells. A better understanding of duct cells is now possible because of the development of techniques for their isolation and culture free from other cell types. Several such techniques are reviewed.

Biochemical and histochemical characterization of cultured rat and hamster pancreatic ducts.

Pancreatic duct fragments were isolated from rat and hamster pancreas and were cultured in an agarose matrix for up to 8 weeks (rat) or 20 weeks (hamster). The fragments consisted predominantly of duct epithelium, lesser numbers of stromal and atrophied acinar cells, and small numbers of islet cells. Hamster ducts averaged 3 micrograms protein per duct while rat ducts averaged 1 microgram, and the protein:DNA ratio of both types of ducts was less than that of whole pancreas. Estimated average duct yields of 6% (hamster) and 1% (rat) were based on the protein content of the ducts. Duct viability was shown by the incorporation of 3H-thymidine and 3H-leucine into bulk DNA and protein and by autoradiography. gamma-Glutamyl transferase and (Na + K)-ATPase specific activities were slightly elevated while amylase was depressed in the ducts when compared with whole pancreas in both species. gamma-Glutamyl transferase was localized histochemically in both duct epithelium and in surviving acinar tissue, as seen in vivo. Amylase was shown by immunohistochemistry to be present within duct lumina and in atrophied acini and their lumina. Alkaline phosphatase and Mg-ATPase specific activities were elevated in the hamster, but reduced in the rat, when compared with whole pancreas. Hamster alkaline phosphatase and Mg-ATPase were localized by histochemistry to the duct stroma, where these enzymes are not detected in vivo. Carbonic anhydrase was found in the duct epithelium of both species, as in vivo, as well as in the duct stroma, unlike in vivo. Acid glycosaminoglycans, as revealed by alcian blue staining, were found at the apical surfaces and in the lumina of both kinds of ducts. Glutathione-S-transferase and glucose-6-phosphate dehydrogenase were elevated in rat ducts, but not in hamster ducts. The polypeptide compositions of cultured ducts, freshly isolated pancreatic islets, and whole pancreas were compared by one-dimensional sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis. No duct-specific polypeptides were observed; the ducts were characterized mainly by the reduction or absence of polypeptides, including some zymogens, seen in whole pancreas.

Effect of duct obstruction on histology and on activities of gamma-glutamyl transferase, adenosine triphosphatase, alkaline phosphatase, and amylase in rat pancreas.

The effect of pancreatic duct obstruction on the activities of amylase and three nonexocrine pancreatic enzymes was studied in the rat. gamma-Glutamyl transferase (GGTase) activity, which is localized primarily in the plasma membrane of acinar cells, disappeared from the acinar basolateral plasma membrane and declined in specific activity by 80% over a seven-day experimental period. Mg-ATPase, localized primarily in the apical plasma membrane of acinar cells, simultaneously declined in activity in acinar cells but increased in activity in connective tissue. Mg-ATPase specific activity rose 3.5-fold. The histochemical results showed that the ductlike cells resulting from obstruction were derived primarily from acinar cells. Alkaline phosphatase (APase) activity, which is localized in vascular endothelium and the stroma of interlobular ducts, exhibited a dramatic increase in the periacinar, periductal, and interlobular stroma, and specific activity rose 11-fold. Amylase-specific activity declined as did the protein to DNA ratio. Gel electrophoresis showed that the amount of zymogen granule polypeptides declined after duct obstruction, whereas a few other polypeptides increased in amount.

Histidyl-proline diketopiperazine cyclo (His-Pro): identification and characterization in rat pancreatic islets.

Measurements of cyclo (His-Pro) in the pancreas were carried out in the rat by a specific radioimmunoassay. Cyclo (His-Pro)-like immunoreactivity was identified in pancreatic islets with a mean concentration of 2023 pg/mg protein, 88-fold higher than that of the whole pancreas. Cyclo (His-Pro) immunoreactivity from pancreatic extracts was indistinguishable immunologically and chromatographically from synthetic cyclo (His-Pro). Insulin-induced hypoglycemia caused a significant, 53% decrease in pancreatic cyclo (His-Pro) concentrations, and FLA-63, a dopamine beta-oxidase inhibitor, also reduced islet cyclo (His-Pro) concentrations 51%. These data indicate that cyclo (His-Pro) is present in rat pancreatic islets and may play a potential role in modulating pancreatic responses to nutrient and pharmacologic stimuli.

Tissue-specific differences in DNA methylation in various mammals.

The only naturally occurring modified base in vertebrate DNA is 5-methylcytosine. Using a precise high-performance liquid chromatographic analysis of DNA enzymatically digested to deoxynucleosides, we have shown that rats, mice and four types of monkey display tissue-specific as well as species-specific differences in the extent of methylation of their cytosine residues. Several similarities in the patterns of tissue-specific DNA methylation in these mammals and in the previously studied human samples were observed. Compared to most other types of DNA examined, brain and thymus DNAs were hypermethylated which suggests that this hypermethylation is a determinant or a necessary byproduct of mammalian differentiation. In all of the studied rodents and primates, the highly repeated DNA sequence fraction was more methylated than the moderately repetitive or single copy fractions. The tissue-specific differences in overall DNA methylation showed no correlation with what is known about average cell turnover rates nor with the percentage of the genome that is transcribed. Liver regeneration in the rat following partial hepatectomy did not detectably alter 5-methylcytosine levels in liver DNA. A considerable increase in the extent of methylation of total liver DNA was observed during normal development of the rat. The latter phenomenon may be due to a major change in the cellular composition of the liver.