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)

The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxic in vivo. Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2 condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P less than 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of halothane on human and rat hepatocyte cultures. 231 35

Matrix vesicles are membrane-invested vesicles that initiate mineralization in the extracellular matrix of calcifying tissues. The epiphyseal cartilages of young-rat rib bones were divided into the growth zone and the resting zone, followed by the isolation of matrix vesicles after collagenase treatment. Matrix vesicles with both alkaline phosphatase and lactate dehydrogenase were detected in the growth cartilage found in the epiphyseal growth plates of young rabbits [Hosokawa, Uchida, Fujiwara & Noguchi (1988) J. Biol. Chem. 263, 10045-10047], but were not detected in the resting zone. By contrast, and surprisingly, lactate dehydrogenase-containing vesicles without alkaline phosphatase were found in the resting zone, but not in the growth zone. In both the growth and resting zones, isoenzyme patterns of lactate dehydrogenase in the two different vesicles were identical with those of cytosolic lactate dehydrogenase of chondrocytes, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase. The same results as for young-rat rib bones were obtained with the resting and growth cartilages of young-dog and monkey rib bones.
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PMID:Vesicles with lactate dehydrogenase and without alkaline phosphatase present in the resting zone of epiphyseal cartilage. 231 Mar 80

Hepatocytes from adult male Sprague-Dawley rats were isolated by the two-stage collagenase perfusion technique; 1 x 10(6) cells/plate were incubated in primary cell culture in Leibovitz's L-15 medium for 24 hr with or without various concentrations (12.5 to 400 mumol/L) of cardioactive cationic amphiphilic compounds such as propranolol, verapamil, sotalol, atenolol and procainamide. Propranolol and verapamil caused a significant release of lactate dehydrogenase (used as cytotoxic index in this study) in the culture media in a concentration-dependent manner, with LC50 values of 220 +/- 10 and 224 +/- 7 mumol/L, respectively. Atenolol, sotalol and procainamide had no effect on lactate dehydrogenase release. Electron microscopy of the hepatocytes showed that subtoxic concentrations of propranolol (12.5 to 125 mumol/L) and verapamil (12.5 to 100 mumol/L) induced multilamellar inclusion bodies after 24 hr of incubation. The two higher concentrations of propranolol (50 and 125 mumol/L) and 100 mumol/L of verapamil produced a significant decrease in the percentage of volume density of the mitochondria as quantitated by morphometrical analysis. An unusual feature of the electron microscopical changes with propranolol and verapamil was the presence of mitochondria within the multilamellar inclusion bodies. When these two drugs were used together or with subtoxic concentrations of amiodarone or desethylamiodarone, release of lactate dehydrogenase was significantly enhanced. No correlation was evident between the cytotoxic response and the volume density of cellular inclusions in hepatocytes treated with different concentrations of propranolol, verapamil, amiodarone or desethylamiodarone. Sotalol, atenolol and procainamide in concentrations up to 400 mumol/L did not produce any ultrastructural changes in hepatocytes after 24 hr of incubation. These results show that (a) cationic amphiphilic structure per se is not the only requirement for induction of multilamellar inclusions, (b) propranolol and verapamil can induce the formation of multilamellar inclusion bodies and cause a concentration-dependent release of lactate dehydrogenase from hepatocytes and (c) combination of different cationic amphiphiles in subtoxic concentrations can enhance cytotoxicity and increase the volume density of multilamellar inclusions.
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PMID:Cytotoxic interactions of cardioactive cationic amphiphilic compounds in primary rat hepatocytes in culture. 237 84

Primary human hepatocytes were used to study bile salt hepatotoxicity and the hepatoprotective potential of ursodeoxycholate in vitro. Hepatocytes were obtained by collagenase perfusion of healthy human liver tissue and were treated with glycochenodeoxycholate for 24 hr 1 day after plating. Clear signs of cytotoxicity were observed at concentrations of about 100 mumol/L glycochenodeoxycholate. Toxicity was determined by release of alkaline phosphatase, gamma-glutamyl transferase, AST, ALT or lactate dehydrogenase into the culture medium, by measuring DNA synthesis of the cultured liver cells and by testing the viability of the hepatocytes using trypan-blue dye exclusion. Addition of ursodeoxycholate, which by itself proved to be of little toxicity, significantly reduced the hepatotoxic effects of glycochenodeoxycholate: 72% +/- 6% of the cells survived treatment with 500 mumol/L glycochenodeoxycholate alone, but addition of 100 mumol/L ursodeoxycholate increased the survival rate to 87% +/- 4% (p less than 0.05). Moreover, all enzymes tested were secreted at a significantly lower level when ursodeoxycholate was present. Similarly, the cellular DNA synthesis was maintained at significantly higher levels as a result of ursodeoxycholate treatment. We conclude that (a) primary human hepatocytes are a suitable model for studying hepatotoxicity of bile salts in vitro, (b) ursodeoxycholate reduces hepatotoxicity of other bile salts and (c) ursodeoxycholate can act hepatoprotectively by itself (i.e., alteration of the metabolism of other bile salts is not necessarily required).
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PMID:Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. 240 54

The effects of culture duration on primary cultured mouse hepatocyte antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and glutathione) and susceptibility to glucose oxidase (GO)- and hydrogen peroxide (H2O2)-induced cell killing and lipid peroxidation were examined. Membrane fatty acid composition was also evaluated. Adult male B6C3F1/CrlBR mouse hepatocytes were isolated by collagenase perfusion of the liver and cultured on 60-mm plastic dishes in Leibovitz's L-15 medium supplemented with glucose (1 mg/ml), dexamethasone (1 microM), fetal bovine serum (10%, v/v), and gentamicin sulfate (50 micrograms/ml) for 0 hr (freshly isolated cells) to 96 hr. Hepatocyte toxicity (determined by lactate dehydrogenase release and lipid peroxidation) after a 2-hr exposure to GO (0.8-80 micrograms/ml) or H2O2 (1-5 mM) decreased with increased time in culture. This decreased hepatocyte sensitivity to GO and H2O2 toxicity was not related to antioxidant enzyme activity since superoxide dismutase, catalase, and glutathione peroxidase declined during the 96-hr culture period. In contrast, glutathione and vitamin E levels in the cultured hepatocytes rose to 274.9 +/- 8.3% and 220.6 +/- 18.6% of the levels in freshly isolated cells (129.6 +/- 11.5 nmol and 0.10 +/- 0.01 nmol per 10(6) hepatocytes, respectively). The percentage of polyunsaturated fatty acids in hepatocyte phospholipids and triglycerides decreased with culture duration while the percentage of oleic acid increased in esterified and free fatty acid pools after 2 hr in culture. Total fatty acids were not affected by time in culture. These results suggest that the decreased hepatocyte susceptibility to the toxic effects of hydrogen peroxide may have been due to elevations in cellular GSH and vitamin E levels and decreases in membrane polyunsaturated fatty acids. The data also indicate that hepatocytes in primary culture undergo changes in antioxidant levels and fatty acid composition that may affect free radical toxicity at different times in culture.
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PMID:Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity. 278 69

The mechanism of the periportal (p.p.) toxicity of allyl alcohol (AlOH) was investigated in p.p. and perivenous (p.v.) hepatocytes isolated by digitonin-collagenase perfusion. The distinct origin of the cell preparations was confirmed by the p.p./p.v. ratios of alanine aminotransferase (p.p./p.v. = 1.8), lactate dehydrogenase (1.3) and glutamine synthetase (0.10). The activity of alcohol dehydrogenase (ADH) was not markedly different in p.p. and p.v. cells. Both types of cells oxidized AlOH at a high but equal rate of about 3 mumol/(min.g cells). Concomitantly with rapid oxidation of 0.7 mM AlOH, glutathione (GSH) was depleted by about 95% and its secretion was completely inhibited in both cell types. Although the GSH content was partially restored during a subsequent 3-h incubation, cellular ATP and K+ content gradually decreased and the leakage of lactate dehydrogenase increased in both types of cells. However, the p.p. cells tended to resist AlOH in vitro better, probably due to their 26% higher GSH content after preincubation with L-methionine. Altering the partial pressure of oxygen in physiological range had no effect on the toxicity of AlOH. The results are contrary to the suggestions that the p.p. location of AlOH liver injury is caused by higher ADH activity or higher oxygen tension in the p.p. zone. Rather, the regiospecificity of the injury may be due to rapid uptake and oxidation of AlOH in the p.p. region.
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PMID:Allyl alcohol cytotoxicity and glutathione depletion in isolated periportal and perivenous rat hepatocytes. 283 85

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.
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PMID:Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes. 290 50

Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.
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PMID:Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes. 299 54

A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process.
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PMID:A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture. 300 46

Intact periportal (pp) or perivenous (pv) hepatocytes were prepared by digitonin-collagenase liver perfusion. The degree of separation was indicated by significant differences between the pp and pv cells in their activity of the pp markers, alanine aminotransferase (pp/pv = 2.1), gamma-glutamyltranspeptidase (3.4) and lactate dehydrogenase (1.3), and of the pv markers, glutamate dehydrogenase (0.73) and pyruvate kinase (0.81). This pattern was not altered by a 3-day pretreatment with phenobarbital (PB). The hepatocytes isolated from the pv area contained higher activities of microsomal NADPH-cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase and benzo(a)pyrene hydroxylase, and of cytosolic glutathione transferase. Cytochrome P-450 and UDP-glucuronosyltransferase were slightly higher in pv cells. Treatment with PB induced NADPH-cytochrome c reductase, glutathione transferase, cytochrome P-450 and UDP-glucuronosyltransferase but the degree of induction was found to be at least as strong in pp cells as in pv cells. The induction of 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase was clearly more prominent in pp cells. On the other hand, PB reduced the activities of benzo(a)pyrene hydroxylase and alcohol dehydrogenase in both cell types. These results demonstrate by direct enzyme assay of separated cells the dominance of the pv-region for metabolizing drugs in the normal liver. Contrary to several other studies, however, our data indicate that induction by PB occurs panacinarily, i.e., relatively more in the pp region, thus diminishing rather than exaggerating the original pv dominance.
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PMID:Effect of phenobarbital on the distribution of drug metabolizing enzymes between periportal and perivenous rat hepatocytes prepared by digitonin-collagenase liver perfusion. 302 20


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