Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Hepatocytes of the small skate (Raja erinacea) were isolated by collagenase perfusion and evaluated by a variety of functional and morphologic criteria. Cell yield was 1.45 X 10(8) +/- 1.3 X 10(7) cells per isolation, and as long as 8 h after isolation 98% of the hepatocytes excluded Trypan blue and no leakage of lactate dehydrogenase (LDH) or cell associated potassium could be detected. Oxygen consumption averaged 1.6 +/- 0.5 nmol/min/mg cell protein, was not stimulated by 1 mM succinate, and also remained stable for up to 8 h following isolation. However, 2,4,-dinitrophenol (5 X 10(-5) M) produced a 55% increase in oxygen utilization while ouabain, (1 mM) or sodium removal decreased oxygen consumption by 31 +/- 6 and 33 +/- 7%, respectively, indicating that a significant portion of the cells energy utilization is coupled to the activity of plasma membrane Na+, K+-ATPase. Light microscopic studies showed that the individual hepatocytes had diameters of 28 +/- 5 microns and contained large lipid droplets. Electron microscopy revealed groups of three to five cells with normal ultrastructure and tight junctions and desmosomes surrounding a single bile canaliculus. These studies indicate that skate hepatocytes can be isolated in high yield that retain their structural polarity in the form of clusters of cells formed around a single bile canaliculus. These hepatocytes remain morphologically intact and metabolically stable for a prolonged period of time.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterization of a polarized isolated hepatocyte preparation in the skate Raja erinacea. 358 70

Hepatocytes from postnatal and adult mice were isolated by perfusion of the liver with a collagenase-containing bicarbonate buffer. These were allowed to attach to collagen-coated tissue culture dishes and were then examined for their susceptibility to paracetamol toxicity. After an 8 hr incubation in either 0.1 or 1.0 mM paracetamol, the extent of lactate dehydrogenase leakage and depletion of glutathione were similar in hepatocytes from young (1-, 2- and 3-week-old) mice when compared to adult mice. The covalent binding of [14C]-paracetamol to protein was greater in the hepatocytes from young mice. The results indicate that while the amount of reactive metabolites free to react with cellular constituents is greater in hepatocytes from young mice, the amount of damage produced was not different than that found in those from adults.
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PMID:Age-related toxicity of paracetamol in mouse hepatocytes. 370 2

Isolated and cultured human hepatocytes provide a useful model for studies of the liver cell function in man. In vitro studies using human hepatocytes are scarce, due to the limited availability and the lack of suitable methods for storage. In this study, we report the effect of deep freezing storage on the viability, fine structures and albumin synthesis of human adult hepatocytes in classical culture conditions. Hepatocytes were isolated using collagenase perfusion (9 isolations). The cell yield was 4-37 X 10(8) with a viability of 60-87%. Cryopreservation was performed in medium containing 10% DMSO and 20% fetal calf serum using a Cryoson BV-4 programmable freezer (0 degree C for 5 min, followed by a freezing rate of 1.5 degrees C/min for 20 min and 7 degrees C/min for 10 min). The cells were stored for 25-275 days in the liquid nitrogen vapor phase (-150 degrees C). Within 16 h about 80% of viable cells from freshly isolated hepatocytes whereas after cryopreservation, 55% of viable cells as determined by Trypan Blue exclusion before the cryopreservation attached to plastic and survived. Electron microscopy showed well developed tight junctions, structures similar to bile canaliculi. Cell polarity was evident. However, 'bleb' formation, more lipid droplets and lysosomes were found in cryopreserved hepatocytes during a short period after thawing. At the 3rd week, cells detached and died. These changes were associated with increased secretion of lactate dehydrogenase, whereas the albumin secretion dropped (from 10 to 4 micrograms/micrograms DNA), regardless of whether hepatocytes were cultured from fresh preparations or after cryopreservation. These findings suggest the cryopreservation is a useful technique to preserve hepatocytes for in vitro studies. Nevertheless, an improved method is necessary to increase the efficiency of cell seeding after cryopreservation.
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PMID:Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures. 374 87

In vitro studies were carried out on isolated rat hepatocytes to examine further the proposed cytoprotective actions of prostaglandins (PG) using carbon tetrachloride (CCl4) as the toxic agent. Isolated hepatocytes, prepared by collagenase, were cultured in Leibowitz-15 medium. Following preincubation, CCl4 (300 or 150 micrograms/ml) was added to the hepatocytes. Treatment with Indomethacin (INDO), 16, 16-dimethyl-PGE2(PGE2) and prostacyclin (PGI2) was assayed in the cultures. Cell damage was measured by lactic dehydrogenase (LDH) release. 6-keto-PGF1 alpha was measured in the supernatant by direct radioimmunoassay. The results showed PGI2 (30.0 ng/ml) treatment 30 min after CCl4 (300 micrograms/ml) addition to be highly protective (p less than 0.001 versus CCl4 control). PGE2 (3 ng/ml) showed similar protection (p less than 0.001). INDO (2 micrograms/ml) following CCl4 (150 micrograms/ml) demonstrated increased cell death (p less than 0.001). INDO (0.5 micrograms/ml) reduced 6-keto-PGF1 alpha production (p less than 0.05). Low dose ethanol (1.5 micrograms/ml) increased 6-keto-PGF1 alpha production (p less than 0.05). Ethanol (1.5 micrograms/ml), added to stimulate endogenous PG production, was cytoprotective when added prior to CCl4 (p less than 0.01). This protection was suppressed by INDO. Ethanol added after CCl4 was not protective. We conclude that exogenously added PGI2 and PGE2 are cytoprotective in this in vitro model and that endogenous PG production may play a protective role in the initial stages of cellular damage.
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PMID:Cytoprotective effect of prostaglandins on isolated rat liver cells. 388 50

Metabolism of 2,6-diisopropylnaphthalene (2,6-DIPN) was studied in freshly isolated carp hepatocytes with special reference to cytochrome P-450-mediated oxidation. The viability of isolated hepatocytes obtained by use of Ca2+-free and collagenase-containing Hanks buffer was 93%, judging from both trypan blue penetration and lactic dehydrogenase (LDH) leakage. 2,6-DIPN was metabolized to form several oxidized products such as the tertiary hydroxy, the primary hydroxy, and two types of dihydroxy DIPN. From the results of the time course experiments, it was assumed that 2,6-DIPN was hydroxylated primarily on the tertiary and primary positions of the isopropyl group, respectively, and thereafter was converted to tertiary-tertiary and primary-tertiary hydroxylated products. These assumptions are supported by results obtained previously in in vivo and in vitro studies.
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PMID:Studies on the sequence of 2,6-diisopropylnaphthalene metabolite formation using carp hepatocyte. 400 35

A method is described for preparing isolated rat submandibular acini by collagenase digestion followed by mechanical dispersion. As assessed by Trypan Blue exclusion, phase contrast microscopy, ATP content and release of mucins and lactate dehydrogenase, the acini are morphologically and functionally intact. Secretory function of isolated acini was similar to that of intact tissue in terms of time-course, dose dependence and degree of stimulation of mucin release by adrenergic secretagogues. Mucin release was increased to the same extent (approx. 3-4-fold) by either isoproterenol or noradrenaline at a maximally effective concentration (10 microM). Stimulation of mucin release by isoproterenol (10 microM), noradrenaline (10 microM) or adrenaline (10 microM) was inhibited by propranolol (30 microM) but not by phentolamine (30 microM). Isoproterenol (10 microM) increased both 45Ca2+ uptake and efflux from the acini, which was shown to represent a net release of calcium. However, there was a delay (approx. 10 min) in onset of stimulation of 45Ca2+ mobilization which was not apparent in isoproterenol stimulation of mucin release. Our results indicate that increases in intracellular calcium mobilization in response to a beta-adrenergic secretagogue do not trigger mucin secretion from rat submandibular acini.
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PMID:Mucin release and calcium fluxes in isolated rat submandibular acini. 609 20

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

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

In order to determine the least injurious method of cell isolation, effluent perfusates from isolated rat hearts were examined for lactate dehydrogenase activity during cellular isolation procedures. Correlation of these results with the perfusates, with modifications in the perfusates, and with the yield of intact cells isolated, indicated that perfusion of isolated hearts with solutions containing bovine serum albumin and/or collagenase can result in severe cellular injury. These effects were significantly modified by the presence of calcium ion. These results indicated that the concentration of calcium ion during isolation is critical to successful isolation of calcium tolerant, functional adult heart cells.
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PMID:Release of lactate dehydrogenase during isolation of adult rat heart cells. 625 64

The response of the rat lung to a range of doses of quartz at 50 and 100 days after its administration by intratracheal instillation has been assessed by bronchopulmonary lavage. The effects on the number of polymorphonuclear leukocytes (PMN), lymphocytes and macrophages are described. In addition the concentrations of soluble protein and hydroxyproline and the activities of lactate dehydrogenase, PZ peptidase and collagenase in lavage fluid supernatants were measured and an assessment of the hydroxyproline content of recovered cells was made. Finally PZ peptidase and collagenase were assayed in PMN-enriched cell fractions and in samples obtained from short-term culture of recovered macrophages. There was a dose-dependent increase in the recovery of all three cell types, and in the amounts of lactate dehydrogenase, protein and hydroxyproline in lavage fluids, which showed no signs of resolution over the 100-day period studied. Measurements of PZ peptidase and collagenase suggested that the PMN, not the macrophages, are the major source of these degradative enzymes. The relevance of these findings with regard to the importance of PMN in quartz-induced fibrosis is discussed.
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PMID:Evidence for a dose-dependent inflammatory response to quartz in the rat lung and its significance in early changes in collagen metabolism. 631 54


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