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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for culturing non- or slowly growing, differentiated fetal rat liver cells is described. It involves the use of collagenase as a digesting agent and of a selective medium deficient in arginine which suppresses the growth of nonparenchymal liver cells. Evidence is presented that surviving cells (a) retain liver-specific urea cycle functions measured by their capacity to transform ornithine into arginine, (b) synthesize DNA in glucose-deficient medium, and (c) synthesize and secrete albumin. This primary cell culture responds to partially hepatectomized rat serum and may be an appropriate assay system for the study of mechanisms which regulate liver regeneration.
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PMID:Studies on primary cultures of differentiated fetal liver cells. 433 10

Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.
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PMID:Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. 435 60

Tenoxicam, a new non-steroidal anti-inflammatory drug has been compared with piroxicam and indomethacin in a range of pharmacological and biochemical inflammation test systems. In a chronic (17-day) adjuvant arthritis in the rat, tenoxicam and piroxicam were equally effective in reducing several indices of inflammation and were less ulcerogenic and better tolerated than indomethacin. The oxicams reduced the oedematous and cellular components of a carrageenan pleurisy at 4 hours while at 24 hours they increased exudate volume and selectively inhibited the accumulation of mononuclear cells. These agents also reduced the inflammatory component of a delayed hypersensitivity response to methylated bovine serum albumin in the mouse. The oxicams were about 100-fold less active than indomethacin as inhibitors of prostaglandin synthetase but all three compounds reduced about equally the release of prostaglandin E2 from phagocytosing rat PMN and interleukin 1-stimulated human rheumatoid synovial cells. The compounds had no effect on the release of superoxide anion, lysosomal enzymes or collagenase from cultured cells, neither did they inhibit isolated collagenase. Only indomethacin stabilized albumin against heat denaturation.
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PMID:Pharmacological and biochemical activities of tenoxicam (Ro 12-0068), a new non-steroidal anti-inflammatory drug. 609 94

The effects of dexamethasone on multiple metabolic functions of adult rat hepatocytes in monolayer culture were studied. Adult rat liver parenchymal cells were isolated by collagenase perfusion and cultured as a primary monolayer in HI/WO/BA, a serum free, completely defined, synthetic culture medium. Cells inoculated into the culture medium formed a monolayer within 24 hr. Electron microscopy showed that the cells in primary culture had a fine structure identical to liver parenchymal cells in vivo, including the observation of desmosomes and bile canaliculi in intercellular space. There was significant gluconeogenesis by the cells 24 hr postinoculation but it had decreased markedly by 48 hr. There was a marked induction of tyrosine aminotransferase (TAT) by dexamethasone, which was maintained for up to 72 hr postinoculation of cells. The transport of alpha-aminoisobutyric acid into the cells in monolayer culture was stimulated by dexamethasone and was dependent on the concentration of dexamethasone. Albumin synthesis and secretion by the cells was measured by a quantitative electroimmunoassay. Albumin production was shown to increase linearly over an incubation period of 24 to 48 hr postinoculation. Dexamethasone depressed the albumin synthesis. The effects of dexamethasone are slow, and at times require more than 6 hr to show variation from the control, indicating that dexamethasone is not a single controlling hormone. Possibly it functions in a cooperative and coordinating role in the regulation of cell metabolism.
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PMID:The effects of dexamethasone on metabolic activity of hepatocytes in primary monolayer culture. 610 97

Primary cultures of liver cells isolated from adult rats by trypsin and collagenase perfusion techniques were carried out to compare cytologic and biochemical properties between the differently prepared cells. Trypsin-dispersed cells consisted of comparatively smaller cells, whereas collagenase-dispersed cells consisted of larger cells. The cell attachment efficiency on culture day 1 was about twice as high in the liver cells prepared with collagenase than those prepared with trypsin. Mature hepatocytes isolated by collagenase perfusion could be maintained in the primary culture for a longer period than those isolated by trypsin perfusion. Epithelial-like clear cells started to grow much earlier in the primary culture of the trypsin-dispersed liver cells than in that of the collagenase-dispersed liver cells. Earlier proliferation of epithelial-like clear cells could not be induced by in vitro trypsinization of the collagenase-dispersed liver cells. Both kinds of enzymatically prepared liver cells showed albumin production and exhibited glucose 6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9, G6Pase) and tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate amino-transferase, EC 2.6.1.5, TAT) activities for 1 week in the primary culture. Albumin production was higher in the liver cells prepared with collagenase than those prepared with trypsin, whereas G6Pase activity was almost the same between them. TAT activity up to culture day 2 was about 3-fold higher in the liver cells prepared with collagenase than in those prepared with trypsin. Combined supplementation of dexamethasone (1 X 10(-5)M) and insulin (10 micrograms/ml) consistently improved the cell attachment efficiency and was very effective in the maintenance of mature hepatocytes in both types. Furthermore, these hormones enhanced the albumin production and TAT activity in both types.
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PMID:Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion. 614 85

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.
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PMID:Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities. 615 76

Livers from human fetuses between the 16th and 24th weeks of gestation were dissociated by successive dispase and collagenase digestion followed by two cycles of low-speed differential centrifugation. This improved method recovered approximately 1 X 10(7) cells (90% hepatocytes and 90% viable cells) from 4 g of liver tissue. These hepatocytes were set into primary culture and monolayer granular hepatocytes were obtained within a week. Both albumin and alpha-fetoprotein production was demonstrated in these granular hepatocytes by the immunoperoxidase method for 2 weeks and alpha-fetoprotein production in the culture medium occurred for a week by the single radial immunodiffusion method. The morphological features of the granular hepatocytes could be distinguished from those of the other type of epithelial cells with clear cytoplasm. During the cultivation period, gradual changes from granular to clear hepatocytes with high mitotic activity were found.
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PMID:Morphological and functional studies of human fetal liver cells in primary monolayer culture. 616 44

Hepatocytes were isolated from 1-day and 1,2,3 and 12-week old rat livers by collagenase perfusion and the relative numbers of albumin (ALB) and alpha-fetoprotein (AFP) producers were evaluated using the reverse hemolytic plaque assay. The percentage of ALB producers remained essentially constant to 35% over the 12-week period. In contrast, the percentage of AFP producers varied from 19% at 1 day up to 28% at 1 week and then down to 0.1% at 3 weeks. Moreover, a double identification of secreting hepatocytes, using an adaptation of the plaque assay, demonstrated that AFP producing hepatocytes were also ALB producers. These results are explained in terms of a restricted specialization of differentiating hepatocytes during normal development.
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PMID:Restricted specialization of differentiating hepatocytes in terms of albumin and alpha-fetoprotein production. 616 64

A superfusion technique was developed as a model system for the study of stimulus-secretion coupling in collagenase-dispersed rat pancreatic acinar cells. Cells (10(7)) were combined with a slurry of Biogel P-4 beads and the mixture was decanted into a plastic column (1.5 cm X 8.5 cm) and perfused with Krebs-Ringer. Amylase activity was determined in sequentially collected effusate fractions and used to estimate the secretory rate. Carbachol, carbachol plus dibutyryl cyclic AMP, cholecystokinin-pancreozymin, and the ionophore A-23187 all stimulated a rapid increase in the rate of secretion. Cell integrity was unaffected by these stimulants as evidenced microscopically and by the lack of lactate dehydrogenase activity in the effusates. Enzymes secreted in response to secretagogues were collected, concentrated, and isoelectrofocused on polyacrylamide gels. A film detection technique was developed to localize amylase activity. The model system has the following advantages: (1) secreted proteolytic products are removed from the vicinity of cells, thereby preventing direct cellular damage and hydrolysis of peptide agonist; (2) the need to add trypsin inhibitors is eliminated and only a minimal addition of albumin (0.001%) is required, thus allowing the separation and distortion-free analysis of secreted proteins; (3) the perfusion conditions can be changed rapidly without disturbing the cells. The model described is therefore well suited to the study of both molecular and kinetic events involved in the enzyme secretory phenomenon in exocrine pancreas.
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PMID:A model system for the study of stimulus - enzyme secretion coupling in rat pancreatic acinar cells. 616 55

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1 g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radioimmunoassay. The capacity of this cell type to store 35S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up 99mTC-colloid sulfur and to incorporate 59Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.
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PMID:The relationship between cell volume, ploidy. and functional activity in differentiating hepatocytes. 617 18


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