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)

A method for the isolation of intact viable rainbow trout liver cells in high numbers is described. The technique involves perfusion of collagenase through the liver. A major part of the cytochrome P-450 in isolated liver cells was present in the oxidized non-substrate bound form. It was observed that 7-ethoxycoumarin was rapidly taken up by the liver cells and bound to cellular cytochrome P-450. The substrate binding spectrum for isolated trout liver cells was slightly modified compared with that obtained with trout liver microsomes. The microsomal affinity of 7-ethoxycoumarin, calculated as the apparent spectral dissociation constant (ks), was elevated 11-fold after fish were treated with beta-naphthoflavone, indicating a qualitative alteration in the nature of the constitutive cytochrome P-450. The metabolism of 7-ethoxycoumarin in isolated liver cells was found to be of a comparable rate to that obtained in liver microsomes. Pretreatment of fish with Clophen A50 or beta-naphthoflavone significantly increased the content of cytochrome P-450 and elevated the rate of 7-ethoxycoumarin deethylation in isolated liver cells. Furthermore, the rate of conjugation of 7-hydroxycoumarin was significantly elevated in liver cells isolated from beta-naphthoflavone treated fish when compared with the control rate. In isolated liver cells, 90% of the 7-hydroxycoumarin formed from deethylation of 7-ethoxycoumarin was further metabolized to conjugated products. However, in beta-naphthoflavone of Clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactions in the cell.
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PMID:Spectral properties of substrate-cytochrome P-450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout liver cells. 399 55

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

The metabolism of 1,3-cyclohexadiene by hepatocytes from phenobarbital induced rat has been investigated. Parenchymal cells were obtained by liver perfusion with a hyaluronidase-collagenase mixture. The addition of the diene to a suspension of hepatocytes gave rise to a type I difference spectrum indicating the formation of an enzyme-substrate complex with cytochrome P-450. The subsequent metabolic pathway of 1,3-cyclohexadiene has been shown to involve, as the first step, the formation of 1,2-epoxy-3-cyclohexene, which is rapidly hydrolyzed to trans-3-cyclohexene-1,2-diol and trans-2-cyclohexene-1,4-diol by a non-enzymatic process. The monoepoxide could not be detected in the incubation medium because of its high reactivity. Therefore, kinetic parameters of the epoxidation reaction were determined by following the rate of production of the diols. When incubated with hepatocytes, trans-3-cyclohexene-1,2-diol, the main product of 1,3-cyclohexadiene metabolism, elicited a reverse type I spectrum, indicating that this compound is not a good substrate for the monooxygenase system.
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PMID:Metabolism of 1,3-cyclohexadiene by isolated rat liver cells. 663 3

Hepatocytes of adult male rats were isolated by hepatic perfusion with a mixture of collagenase and hyaluronidase. This procedure gave a high yield of viable cells, as determined by trypan blue exclusion. The addition of 1,3-cyclohexadiene to a suspension of isolated liver cells gave rise to a type-I spectral change indicative of the formation of the cytochrome P-450-substrate complex. 1,3-cyclohexadiene was then incubated with hepatocytes for different times, to determine its biotransformation. Cyclohexadiene-1,2-diol resulted to be the main metabolite. It is proposed that the metabolic pathway of 1,3-cyclohexadiene involves the intermediate formation of an epoxide which is rapidly transformed to the correspondent diol.
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PMID:[Preliminary results on the metabolism of 1,3-cyclohexadiene in isolates rat hepatocytes]. 706 93

Rat hepatocytes were isolated by a collagenase perfusion technique with subsequent subfractionation on Metrizamide gradients into subpopulations which have been designated band I and band II and are likely to be enriched with centrilobular and periportal cells, respectively. Band I was found to have a higher concentration of 5'-nucleotidase and band II a higher concentration of alcohol dehydrogenase. Furthermore, pretreatment of rats with phenobarbital led to higher cytochrome P-450 in the band I (centrilobular enriched) as compared to the band II (periportal enriched) subpopulations of hepatocytes. These data support their ascribed lobular origins. The uptake of a single concentration of galactose, ouabain and taurocholate into each of the two subpopulations was investigated until the concentration within the hepatocytes no longer increased. No difference was found in the uptake of [14C]galactose (25 mM) between the two hepatocyte subpopulations. However, the uptake of [3H]ouabain (125 microM) was greater in the centrilobular as compared to periportal enriched fraction of the hepatocytes. An even greater difference was found for the uptake of [3H]taurocholate (25 microM). The kinetics of taurocholate uptake were subsequently investigated. The Km for each subpopulation was 21 microM, while the Vmax of the centrilobular enriched fraction was 2.03 and that of the periportal enriched fraction was 1.57 nmol/min/mg of protein. These results show that there is a difference in uptake into hepatocytes of centrilobular and periportal origin for ouabain and taurocholate, but not for galactose.
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PMID:Uptake of galactose, ouabain and taurocholate into centrilobular and periportal enriched hepatocyte subpopulations. 720 41

Primary cultures of adult rat parenchymal hepatocytes were developed as an in vitro model to investigate the biochemical fate of 2-acetylaminofluorene (AAF), a potent hepatocarcinogen. More than 5 x 10(8) viable cells were routinely isolated by collagenase perfusion in rat liver; the cells were cultured 2-5 d on collagen-coated dishes in serum-free culture medium containing hormones and other factors to retard the decline of cytochrome P-450. All of 137 ng or 13.7 microgram AAF was metabolized in 21-24 h by 2 x 10(6) cultured hepatocytes in 4.0 ml defined medium. At the higher dose, water-soluble metabolites appeared at 70% of the rate of metabolism at the lower dose, which was 17 ng/h for the initial 4 h. As the parent compound was consumed, bound AAF residues were recovered with exhaustively extracted, trichloro-acetic acid-precipitated hepatocellular macromolecules, accounting for a maximum of 5% of the 137-ng dose. Addition of hormones to the culture medium stimulated the rate of appearance of water-soluble metabolites, AAF, correlating with the enhanced cytochrome P-450 levels of hormone-treated cells. Metabolism of AAF was diminished 50% during 3 h of incubation with 10(-4) M SKF 525A and 100% with 10(-3) M SKF 525A. At a dose of 40 microgram AAF per 2 X 10(6) cells, only 31% of the carcinogen was recovered from the culture medium as water-soluble products after 24 h; the cells were sown to be capable of metabolizing a subsequent 40-microgram dose at an undiminished rate, suggesting that saturation of metabolizing enzymes rather than toxicity occurred. These results support the validity of primary hepatocyte cultures as a model system for quantitative investigations of the biochemical fate of AAF in mammalian cells, and provide preliminary characterization of the cells' processes of detoxification and metabolic activation of a chemical carcinogen.
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PMID:Metabolism of 2-acetylaminofluorene in primary rat hepatocyte cultures. 726 2

Current bioartificial liver devices are based on the use of a large mass of hepatocytes exhibiting differentiated metabolic function. The pig has become a source of interest for the acquisition of such cells-however, harvesting a large mass of highly viable cells has met with difficulty. This study describes a technique for harvesting large quantities of hepatocytes at viabilities greater than 90% and also describes several features documenting differentiated function. Pigs, 6 to 10 kg body weight, underwent in situ two-step whole liver perfusion (ethylene glycol tetraacetic acid and collagenase) and ex vivo cell harvest. Harvests yielded an average of 19.5 billion cells with an average viability of 94.6%. Hepatocytes were then entrapped in type I collagen (3 x 10(5) cells/well) and cultured in serum-free media for 5 days. Pig hepatocytes produced stable amounts of albumin and maintained cytochrome P-450 and glucuronidation activity over 5 days, as shown by the metabolism of lidocaine and 4-methylumbelliferone. These data indicate that pig hepatocytes can be harvested with high yields and can retain viability and differentiated function over at least 5 days of culture, and therefore should prove to be an excellent source of hepatocytes for bioartificial liver devices.
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PMID:A technique for porcine hepatocyte harvest and description of differentiated metabolic functions in static culture. 777 Sep 34

The abundance of 12 cytochrome P-450 (CYP) mRNAs was quantified in the caudate lobe of rat livers before dissociation of the organ into single cells by perfusion with 0.025% (w/v) collagenase. Comparison of the initial abundance of CYP-1A1, -1A2, -2A subfamily, -2B1/2, -2C7, -2C11, -2D subfamily, -2E1, -3A1/2 and -4A1 transcripts in the caudate lobe of the intact liver with the values found in freshly isolated hepatocytes demonstrated that the relatively brief (1 h) cell isolation and washing procedures routinely caused 2-3-fold increases in the mRNAs encoding CYP-1A2, -2B1/2, -3A1/2, and -4A1, concomitant with a 50% decline in CYP2C11 mRNA. Further changes in the expression of CYP mRNAs occurred when the hepatocytes were cultured. Thus CYP1A1 mRNA, which is not constitutively expressed in rat liver, became detectable in hepatocytes cultured for 1 h, and after 6 h CYP3A1/2 mRNA levels began to increase. In contrast, levels of all other CYP mRNAs studied had declined after 24 h of culture concomitant with the loss of total cytochrome P-450 content. Culture of hepatocytes with 0.5 mM metyrapone (which prevents the loss of total P-450 content) increased CYP1A1 and CYP3A1/2 mRNA levels still further, such that after 72 h of culture these transcripts were conservatively 10-18-fold higher than in hepatocytes prior to culture. This suggests that these two isoenzymes comprise the bulk of the total cytochrome P-450 content maintained by metyrapone. Collectively, these results demonstrate that the technique commonly used to isolate rat hepatocytes alters hepatic gene expression, as illustrated by the elevation of the mRNAs encoding CYP-1A2, -2B1/2, -3A1/2 and -4A1, and that such perturbations are exacerbated during culture under standard conditions by the loss of the constitutive CYP2C11 and the precocious induction of CYP1A1 and CYP3A1/2 mRNAs.
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PMID:Altered expression of cytochrome P-450 mRNAs, and potentially of other transcripts encoding key hepatic functions, are triggered during the isolation of rat hepatocytes. 843 60

Morphologically and functionally intact human hepatocytes were isolated from small liver biopsy samples weighing about 1-2 g by initial digestion with collagenase followed by repeated digestions with trypsin. The usual yield of hepatocytes was greater than 1 x 10(7) cells per g of liver sample and cell viability, as judged by dye exclusion test, was routinely over 90%. The isolated human hepatocytes showed intact morphology under scanning electron microscope. Formation of membrane protrusions upon phalloidin addition demonstrated that the actin in isolated hepatocytes was maintained with its structural integrity. The cultured human hepatocytes retained a variety of liver-specific functions which were similarly exhibited by rat hepatocytes isolated using the same procedure. The cultured human hepatocytes exhibited a specific cytochrome P-450 related enzyme activity, and active amino acid uptake that increased upon addition of hormones like glucagon and dexamethasone. Additionally, the cultured human hepatocytes synthesized DNA actively and, human serum albumin, and was found to be responsive to modulation by growth modulating hormones, cytokines and hepatotoxic agents. Based on the profile of activity described above, the presently established conditions for isolation and culturing of human hepatocytes demonstrate that functional liver cells can be obtained from small biopsied liver samples.
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PMID:Functional human hepatocytes: isolation from small liver biopsy samples and primary cultivation with liver-specific functions. 872 Jan 63

Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and ethylmorphine starting with either N-demethylation or O-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metabolism of ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total ethylmorphine elimination rate was reduced by 12% at 5mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for ethylmorphine-->norethylmorphine, 0.63 for norethylmorphine-->normorphine, 0.56 for ethylmorphine-->morphine, 0.49 for morphine-->normorphine, 0.31 for normorphine-->normorphine-3-glucuronide and 0.49 for morphine-->morphine-3-glucuronide. Almost similar effects of ethanol on codeine metabolism were found. In additional experiments, norethylmorphine or norcodeine (50 microM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norethylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and normorphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation process were the ones most affected by ethanol.
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PMID:Effects of ethanol on ethylmorphine metabolism in isolated rat hepatocytes: characterization by means of a multicompartmental model. 914 Jan 36


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