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)

We recently found that polyunsaturated lecithin prevents ethanol from causing cirrhosis in the baboon. Because transformation of lipocytes to transitional cells plays a key role in hepatic fibrogenesis in vivo, and because this process in alcohol-fed baboons was found to be attenuated by polyunsaturated lecithin, we focused on lipocytes to study the mechanism of the protective effect. Rat lipocytes cultured on plastic undergo spontaneous activation, accompanied by expression of alpha-smooth muscle actin isoform and production of substantial amounts of type I collagen. The latter was further increased on incubation with acetaldehyde. This in vitro model was used here to study how acetaldehyde-mediated collagen production and accumulation can be turned off. Addition of polyunsaturated lecithin (10 mumols/L) was found to prevent the acetaldehyde-induced increase in collagen accumulation by 83% (p less than 0.001). By contrast, a saturated phospholipid (10 mumols/L dilauroyl phosphatidylcholine), a monounsaturated one (10 mumols/L linoleoyl-palmitoyl phosphatidylcholine) or linoleic acid (20 mumols/L bound to albumin) had no such effect. Incorporation of [3H]proline into collagen and the expression of alpha-1 (I) procollagen mRNA were increased by acetaldehyde; the latter was not significantly affected by polyunsaturated lecithin. Polyunsaturated lecithin increased lipocyte collagenase activity by 100% (p less than 0.001), whereas dilauroyl phosphatidylcholine, linoleoyl-palmitoyl phosphatidylcholine and linoleic acid had no such action. We concluded that (a) polyunsaturated lecithin selectively prevents the acetaldehyde-induced increase in collagen accumulation in lipocyte cultures, whereas other phospholipids or linoleate have no such effect; and (b) polyunsaturated lecithin does not modify the acetaldehyde-mediated increase in alpha-1 (I) procollagen mRNA, but it increases collagenase activity, suggesting that the protective effect exerted by polyunsaturated lecithin against alcohol induced fibrosis in vivo is due at least in part to stimulation of collagenase activity, which may prevent excess collagen accumulation by offsetting increased collagen production.
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PMID:Polyunsaturated lecithin prevents acetaldehyde-mediated hepatic collagen accumulation by stimulating collagenase activity in cultured lipocytes. 137 80

In addition to fibrosis in response to necrosis and inflammation, alcohol may promote fibrogenesis directly, resulting in pericellular, perisinusoidal and perivenular fibrosis, in association with increased collagen mRNA. Acetaldehyde (produced in increased amounts because of the selective induction of cytochrome P450IIE1) stimulates collagen formation from either myofibroblasts, Ito cells or fibroblasts. One postulated mechanism is adduct formation of acetaldehyde with intracellular proteins, possibly stabilized by the NADH generated upon ethanol oxidation. The latter also increases lactate which might inhibit proline oxidase activity and increase available proline. During the initial stage of alcohol consumption, collagenase activity is increased, in keeping with enhanced collagen production. In later stages, however, there is a secondary decrease of collagenase activity; its deficiency relative to synthesis may also promote collagen deposition. In the early stage of alcohol induced fibrosis, collagens type I and type III accumulate, whereas later, type I predominates. Both can be formed in vitro by Ito cells, but cultured hepatocytes produce mainly type III. Immunohistochemical techniques revealed the deposition of type III procollagen in the extrahepatic collagen fibrils. Its degradation, as well as its enhanced synthesis, contribute to the appearance of procollagen III peptides in the blood. Their measurement by Fab fragments of the antibody is useful to assess the degree of fibrosis and to detect perivenular fibrosis, a precirrhotic lesion. In the baboon model, polyunsaturated lecithin was found to be effective in opposing alcohol-induced fibrosis.
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PMID:Alcohol and fibrogenesis. 184 59

We tested the effect of ethanol and its metabolite, acetaldehyde, on bone formation as measured by [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagen protein (NCP), and on DNA synthesis as measured by [3H]thymidine (TdR) incorporation in fetal rat calvaria. We also determined the effects of ethanol and acetaldehyde on prostaglandin E2 (PGE2) release from calvaria and on bone resorption as measured by 45Ca release from fetal rat long bones. Bones were cultured in multiwell plastic dishes (open system) or in stoppered Erlenmeyer flasks (closed system) for 24 to 96 h. In the open system, 1% ethanol (v/v; 172 mM) resulted in a 31% decrease in TdR incorporation at 24 h with no effect on CDP and NCP. At 0.1% (17.2 mM), ethanol increased TdR by 22%, CDP by 73% and NCP by 67% at 24 h, but these effects were not sustained at 96 h. At 24 h, 1% and 0.3% ethanol decreased PGE2 release by 88% and 75% respectively. This effect was sustained for 96 h only at the higher concentration. In the closed system, 0.1% ethanol increased TdR incorporation by 38% at 24 h. However, there was no effect on the labeling of CDP or NCP. Because its boiling point is 21 degrees C, acetaldehyde could only be tested in the closed system. Acetaldehyde markedly inhibited bone metabolism. At 24 h, 0.003% (0.54 mM) to 0.01% (1.79 mM) acetaldehyde caused a dose-related inhibition of TdR incorporation from 23 to 45%. At 0.01% and 0.03% acetaldehyde inhibited proline incorporation into CDP by 48% and 94% and NCP by 40% and 74% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of ethanol and acetaldehyde on collagen synthesis, prostaglandin release and resorption of fetal rat bone in organ culture. 233 31

Periportal and perivenous hepatocytes were isolated by the digitonin-collagenase perfusion technique. The activity of the cytosolic glutathione S-transferase was higher in perivenous cells, but the cytosolic glutathione reductase and the microsomal glutathione S-transferase activities were evenly distributed. In contrast, both the Se-dependent and the microsomal Se-independent glutathione peroxidase activity and the glucose-6-phosphate dehydrogenase activity was much lower in perivenous hepatocytes, suggesting that these cells have a lowered detoxification capacity, which may contribute to their greater susceptibility to damage by xenobiotics. The mechanism of the ethanol-induced GSH depletion in vivo was studied by incubating conventionally isolated hepatocytes. In the absence of glutathione precursors, ethanol (80 mM) did not influence the GSH content, despite accumulation of acetaldehyde (10-100 MicroM). L-Methionine or L-cysteine stimulated GSH replenishment to in vivo rates. Ethanol oxidation resulted in acetaldehyde accumulation, but did not inhibit GSH replenishment from L-methionine and even stimulated that from L-cysteine. This seems to exclude conjugation of GSH with acetaldehyde as a mechanism by which ethanol suppresses GSH levels in vivo.
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PMID:Glutathione metabolism in isolated rat hepatocytes: acinar heterogeneity of detoxifying enzymes and effects of ethanol. 342 86

Acetaldehyde, the first metabolite of ethanol, mediates many of the biological effects of ethanol. We have previously shown that acetaldehyde, but not ethanol, stimulates collagen production in cultured human fibroblasts (Holt, K., Bennett, M., and Chojkier, M. (1984) Hepatology 4, 843-848). Here, we examined the effects of acetaldehyde on collagen gene expression. Confluent human fetal fibroblasts were incubated for up to 4 h in the presence of ascorbate (0.2 mM) alone or with the addition of either ethanol (12 mM) or acetaldehyde (200 microM). Acetaldehyde induced the production of collagen (up to 2.5-fold) and had a small inhibitory effect on procollagen secretion (-20%). The steady-state levels of mRNAs were measured by hybridizing total cellular RNA to specific cDNA probes at high stringency. Acetaldehyde increased the steady-state level of collagen alpha 1(I) and collagen alpha 2(I) mRNAs about 3-fold and had small effects on beta-actin mRNA (+50%) and collagenase mRNA (-50%). Northern blots revealed that the RNAs were intact and that acetaldehyde preferentially increased the abundance of the longer of the two collagen alpha 1(I) transcripts. Acetaldehyde increased both collagen alpha 1(I) and collagen alpha 1(III) transcriptional activity by 2.5-fold and had small effects on beta-actin and collagenase gene transcription. The increase in both collagen production and collagen mRNA levels induced by acetaldehyde was blocked by methylene blue, a scavenger of reducing equivalents. These data indicate that reducing equivalents, which enhance the formation and stability of acetaldehyde-protein adducts, may be required for acetaldehyde-stimulated collagen production. Thus, this study suggests that acetaldehyde increases collagen production by increasing collagen gene transcription in cultured human fibroblasts.
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PMID:Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. 369 68

Ethanol metabolism in rat hepatocytes isolated either from the periportal (pp) or the perivenous (pv) area by collagenase gradient perfusion was compared to reveal metabolic factors that could be associated with the development of perivenous alcoholic liver damage. Cells were also isolated from rats given ethanol (E) chronically by addition to the drinking fluid. One group (EM) received in addition the alcohol dehydrogenase inhibitor 4-methylpyrazole, which potentiated the ethanol treatment by causing sustained elevated diurnal blood ethanol levels. Fatty degeneration ensued in only one-third of the E rats but in all of the EM rats. The periportal/perivenous activity distributions of alanine aminotransferase (ALAT) and glutamate dehydrogenase (GLDH) were 2.2 and 0.75, respectively. Both ethanol treatments significantly decreased the ALAT and increased the GLDH activities, but did not change their pp/pv distributions. Ethanol treatment also increased ethanol and acetaldehyde oxidation, but to the same extent in pp and pv cells. The increase was more marked in cells from EM rats despite their more severe liver fatty degeneration. Ethanol incubation also increased the lactate/pyruvate ratio to the same extent in pp and pv cells both from control or ethanol-treated rats. Our results indicate that periportal and perivenous hepatocytes convert ethanol via acetaldehyde to acetate equally well and with similar effects even after chronic ethanol treatment. Consequently, preferential damage of the perivenous area after chronic ethanol intake is not caused by inherent or acquired differences in ethanol metabolism between perivenous and periportal hepatocytes. Rather, sinusoidal gradients only established in the intact liver may exaggerate the metabolic imbalance by ethanol in the perivenous area, thus explaining its greater vulnerability to damage by alcohol abuse.
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PMID:Comparison of ethanol metabolism in isolated periportal or perivenous hepatocytes: effects of chronic ethanol treatment. 390

Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.
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PMID:Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts. 638 Dec 14

The mechanisms responsible for the increased hepatic collagen deposition in alcoholic cirrhosis remain unknown. The question of whether ethanol or acetaldehyde has a direct effect on collagen and noncollagen protein production was investigated in human fibroblasts with no detectable activity of alcohol dehydrogenase to distinguish the effects of these metabolites. To eliminate environmental factors, protein production by confluent human skin, fetal and hepatic fibroblasts was studied after three passages. Cells were labeled with [5-3H]proline for 4 hr in the presence of 0.2 mM ascorbate alone or with addition of either ethanol (50 mM) or acetaldehyde (0 to 300 microM). Rates of protein production were calculated from the radioactivities of collagenase-sensitive and collagenase-resistant proteins. Skin fibroblasts from alcoholic individual either with cirrhosis or without liver disease have comparable rates of collagen and noncollagen protein production. Acetaldehyde, in a concentration found in the liver during ethanol abuse, significantly increased collagen production by human skin fibroblasts (up to 140%), fetal fibroblasts (up to 240%) and hepatic fibroblasts (up to 70%) but the addition of ethanol had no significant effect on basal collagen production. The effect of acetaldehyde was dose-related and affected noncollagen protein production in a similar manner. Acetaldehyde did not cause changes in either proline transport or the specific activity of the proline precursor pool. This newly recognized stimulation of collagen production by acetaldehyde may be a possible mechanism of fibrogenesis in alcoholic individuals.
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PMID:Acetaldehyde stimulates collagen and noncollagen protein production by human fibroblasts. 647 53

In an effort to evaluate further the concept of ethanol-induced lipid peroxidation, isolated rat hepatocytes obtained via collagenase perfusion were utilized. Hepatocytes were judged to be functionally intact based on measurements of adenosine-5-triphosphate, gluconeogenesis, bromosulphthalein uptake, and trypan blue exclusion. When hepatocytes were incubated with acetaldehyde, a metabolite of ethanol, at 100 mg% and 10 mg%, significant increases in lipid peroxidation resulted as measured by levels of malonaldehyde. Acetaldehyde-induced increases in malonaldehyde were reduced by pre-incubation with antioxidants such as Vitamin E (200 mg%) or glutathione (100 mg%).
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PMID:Acetaldehyde-induced lipid peroxidation in isolated hepatocytes. 710 Jun 31

Alcoholic and, to a lesser extent, nonalcoholic patients with liver disease have serum antibodies to acetaldehyde-protein adducts produced in vitro. These antibodies presumably reflect the presence of adducts in the liver, but the protein that triggers this immune response has not been identified. To study this, we measured the reactivity of cytosolic proteins to rabbit IgG developed against a P-450 2E1-acetaldehyde adduct, isolated from alcohol-fed rats, that recognizes acetaldehyde-modified epitopes in proteins. Adducts were determined on Western blots by scanning densitometry of antibody-linked alkaline phosphatase activity in 4 normal livers and in needle biopsy specimens from subjects with liver disease, 17 alcoholic and 14 nonalcoholic. In all livers, except for a normal one, we found a reactive protein of at least 200 kD, similar to the collagen-acetaldehyde adduct we reported to be markedly increased in rats with experimentally induced cirrhosis. The immunostaining intensity in the alcoholic patients with liver disease was eightfold (p < 0.01) and that in nonalcoholic patients with liver disease was fourfold, greater (p < 0.02) than the weak staining in normal livers; it correlated with the degree of inflammation and serum AST or gamma-glutamyl transpeptidase activities. The adduct was reproduced on incubation of normal cytosolic proteins with 2.5 mmol/L acetaldehyde, whereas higher concentrations yielded many additional adducts; the adduct also reacted with IgG antibody to rat collagen type I and disappeared after digestion with collagenase, suggesting that the target protein is a form of collagen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Collagen-acetaldehyde adducts in alcoholic and nonalcoholic liver diseases. 791 86


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