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 have developed a fluorometric cytochemical assay for gamma-glutamyltranspeptidase (gamma-GT) using the substrate gamma-glutamyl-4-methoxy-2-naphthylamide in which the released methoxynaphthylamine was coupled with 5-nitrosalicylaldehyde to form a yellow fluorescent crystalline product within the cells. Single cell suspensions were obtained by collagenase perfusion of livers from rats that had either received a two-thirds partial hepatectomy followed 24 hr later by a single injection of diethylnitrosamine (DEN) or received a partial hepatectomy alone. Cultured HTC cells were used as a source of gamma-GT+ cells. Fluorescence (gamma-GT activity) was low in most of the cells from both DEN-exposed and control rats, but high in HTC cells. The livers of both DEN-exposed and control rats had a subpopulation of cells that were gamma-GT+; this population could be quantitated and sorted by flow cytometry. Five weeks post injection the number of GT+ cells from the rats exposed to DEN was more than 20 times that from the control rats. Increased gamma-GT activity may be a useful cytochemical marker for preneoplastic liver cells.
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PMID:Flow microfluorometric identification of liver cells with elevated gamma-glutamyltranspeptidase activity after carcinogen exposure. 3 64

Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated gamma-glutamyltranspeptidase in greater than 95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectin Arachis hypogaea (rat proximal tubule) and negatively for Lotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, gamma-glutamyltranspeptidase and leucine aminopeptidase, and Na(+)-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium.
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PMID:Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury. 171 31

A cell fraction enriched in biliary epithelial cells (BEC) has been isolated from the liver of normal rats. The procedure involved proteolytic digestion by trypsin and mild mechanical disruption of biliary ductular and connective tissue that remained undigested after collagenase-hyaluronidase perfusion. An adherence procedure removed the large majority of contaminating Kupffer cells. The majority (87.4 +/- 3.5%) of the cells were positive to an indirect immunofluorescence staining that used an antiserum against bovine hoof prekeratin that specifically recognizes intermediate filaments of biliary epithelium. Similar results were obtained by histochemical staining for gamma-glutamyltranspeptidase activity. The contamination of the BEC fraction with Kupffer cells and hepatocytes was approximately 7% and 2%, respectively. The viability of the BEC population was always more than 90%. The BEC and hepatocytes were analysed for their lipid composition; the BEC were found to have a cholesterol content approximately 6-times higher than hepatic parenchymal cells, with a cholesterol/phospholipid molar ratio of 0.53 in comparison to a value of 0.11 for hepatocytes. No detectable evidence of cytochrome P-450 or cytochrome P-450-related enzymatic activities was found in the BEC.
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PMID:Isolation and characterization of biliary epithelial cells from normal rat liver. 245 9

Hepatocytes were aseptically isolated from either the periportal (pp; zone 1) or the perivenous (pv; zone 3) region by digitonin-collagenase perfusion and cultured on type I collagen for 4 to 9 days. In freshly isolated cells the pp:pv activity ratios of the acinar marker enzymes gamma-glutamyltranspeptidase (gamma-GT), alanine aminotransferase (ALAT) and glutamate dehydrogenase (GLDH) were 2.8, 1.6 and 0.76, respectively. During culture ALAT and GLDH activities gradually declined, but the pp-pv difference was retained for at least 4 days. In contrast, the difference in the gamma-GT activity was rapidly lost, due to its fast initial activation in pv cells. The initial 7-ethoxycoumarin O-deethylase (ECDE) activity was higher in pv cells; this difference was retained for several days of culture and was increased by induction in vitro with either phenobarbital (PB) or beta-naphthoflavone (beta NF). Although the basal UDP-glucuronyltransferase (UDPGT) activity with either p-nitrophenol (pNP) or hydroxybiphenyl (HBP) as substrate did not differ significantly, the in-vitro PB- or beta NF-induced activity was higher in pv cells. Both glucuronidation and sulfation of methylumbelliferone tended to be higher in pv cells. Glutathione S-transferase was initially significantly higher in pv cells and this difference was augmented after in vitro induction by PB or beta NF. After six days in culture all the observed pp-pv differences had disappeared. These results suggest that hepatocytes isolated from the perivenous region seem to maintain their initially higher capacity for phase I and phase II drug reactions during culture and also respond more strongly than periportal cells to in vitro induction.
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PMID:Drug metabolism by periportal and perivenous rat hepatocytes. Comparison of phase I and phase II reactions and their inducibility during culture. 256 12

Sequential carcinogen treatment (diethylnitrosamine/partial hepatectomy followed by 2-acetylaminofluorene (2-AAF] induced multiple hepatocarcinomas in rats with 100% certainty within a year. Enzyme-altered lesions, i.e. gamma-glutamyltranspeptidase (GGT)-positive and/or ATPase-negative cell foci, were numerous already at 8 weeks, and suspensions of purified hepatocytes isolated (by collagenase perfusion) at this time contained 30-40% GGT-positive cells. These hepatocyte suspensions were markedly deficient with respect to autophagic protein degradation (in comparison with cell suspensions from normal rats), and the cells lost less protein and survived much better than normal hepatocytes in culture under conditions of amino acid deprivation (which activates the autophagic mechanism). The anabolic advantage of reduced autophagy may possibly contribute to the selective outgrowth of preneoplastic cells during the earliest stage of liver carcinogenesis. Inclusion of the autophagy inhibitor 3-methyladenine in the culture medium elevated the survival of normal hepatocytes up to the level seen with hepatocytes from carcinogen-treated animals, suggesting that protection of normal cells by autophagy suppression may be a potentially interesting therapeutic principle.
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PMID:Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. 285 48

On treatment with collagenase, brain microvessels, together with several protein components, lose some enzymatic activities such as alkaline phosphatase and gamma-glutamyltranspeptidase, whereas no change occurs in the activities of 5'-nucleotidase and glutamine synthetase. The energy-requiring "A-system" of polar neutral amino acid transport is also severely inactivated, whereas the L-system for the facilitated exchange of branched chain and aromatic amino acids is preserved. In the collagenase-digested microvessels, this leads to loss of the transtimulation effect of glutamine on the transport of large neutral amino acids, because such transtimulation is due to a cooperation between the A- and L-systems. By contrast, NH4+ maintains (and even enhances) its ability to stimulate the L-system of amino acid transport, presumably through glutamine synthesis within the endothelial cells.
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PMID:Isolated brain microvessels as in vitro equivalents of the blood-brain barrier: selective removal by collagenase of the A-system of neutral amino acid transport. 289 Jul 11

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

Suspensions of freshly isolated hepatocytes were prepared by collagenase perfusion of livers of adult Fischer 344 female rats. The cells were injected into the dorsal fascia of 2/3 partially hepatectomized syngeneic hosts (10(6) cells per injection site) and were monitored from 3 days to 3 months after injection. Brown nodules developed at the transplantation site. Histologic examination of the nodules revealed that the hepatocytes were arranged in cords and clusters surrounded by fibrovascular connective tissue. Bile ductules were also seen. Hepatocytes were positive for glucose-6-phosphatase. Staining for gamma-glutamyltranspeptidase showed that the parenchymal hepatocytes were mostly (approximately 95%) negative, whereas bile ductules were positive. These histochemical findings were seen in hepatocytes up to 3 months after transplantation and did not vary with the age of the transplants. Electron-microscopic examination of the transplanted nodules demonstrated that the cells maintained the characteristics of hepatocellular cytoplasmic structure. The relationship between the bile canaliculi and the stromal vessels was found to be similar to the bile canaliculi and hepatic sinusoid polarity seen in the normal liver. Autoradiographic analysis showed that a fraction of the transplanted cells was active in DNA synthesis. This system may become a tool in the study of survival and neoplastic transformation of hepatocytes as a result of exposure to X-irradiation and chemical carcinogens.
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PMID:Morphologic and histochemical analysis of hepatocytes transplanted into syngeneic hosts. 610 19

Rat pancreases were minced and treated with collagenase or collagenase supplemented with chymotrypsin to yield a mixture of ducts, islets, acinar cell clusters, blood vessels, and nerves. Histologically and ultrastructurally, the isolated tissues resembled their in situ counterparts in most respects, the major difference being the destruction of the basement membranes (basal laminae). Ducts ranging in size from the common bile/main pancreatic duct to the intercalated ducts were identified in the digest, although interlobular ducts were most frequently observed. Acinar tissue fragments were separated from nonacinar structures either by flotation through discontinuous gradients of Ficoll or by sieving, the latter technique being the more efficient. Common bile/main ducts, interlobular ducts, and blood vessels were selected manually from the nonacinar fractions. Biochemical analyses showed that the entire nonacinar fraction, as well as isolated ducts and blood vessels, contained larger alkaline phosphatase, carbonic anhydrase, and Mg-ATPase specific activities than acinar tissue, whereas acinar tissue contained larger gamma-glutamyltranspeptidase and amylase activities. However, greater than 63% of the total recovered activity of each enzyme was associated with the acinar tissue. Both the association of the majority of each of these enzyme activities with the acinar tissue and the similarity in specific activities associated with ducts and blood vessels indicate that none of the enzymes tested is a unique marker for interlobular and larger ducts of the pancreas of the rat.
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PMID:Characterization of ducts isolated from the pancreas of the rat. 615 56

Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model). The isolation procedure involved, after collagenase dispersion of the liver, separation of the hepatocytes into small- and large-cell fractions by centrifugal elutriation, and subsequent selection of cells deficient in asialoglycoprotein receptor(s) by plating onto asialofetuin (ASF)-coated plates. The number of cell surface binding sites for the asialoglycoprotein receptor was measured with both asialoorosomucoid and ASF as ligands. There was a 50% reduction of binding sites for both ligands in the original cell suspensions obtained from preneoplastic livers. The reduction in receptor binding sites was most pronounced in the large cell fraction (less than or equal to 30% of control value) after separating the original cell suspension by elutriation into small and large cell fractions. Immunohistochemical studies showed a lack of asialoglycoprotein receptor in preneoplastic (i.e., hyperplastic foci) areas. These areas were entirely super-imposable with glucose-6-phosphatase-deficient areas and partially overlapped the gamma-glutamyltranspeptidase-positive areas in serial liver sections. The attachment of preneoplastic hepatocytes to ASF-coated tissue culture dishes was greatly impaired, and the number of gamma-glutamyltranspeptidase-positive cells on the ASF dishes was reduced to less than 7% as compared to 45 to 70% on the collagen-coated plates. Thus, the lack of asialoglycoprotein (asialofetuin) surface receptors and the increased size of the early preneoplastic hepatocytes are characteristics that can be used to separate the preneoplastic cell population from normal liver cells.
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PMID:Isolation of preneoplastic rat liver cells by centrifugal elutriation and binding to asialofetuin. 620


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