Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Liver parenchymal cells from adult rats were isolated by treatment with collagenase and cultured as monolayers in Williams medium E with 10% fetal or calf serum. The additions of dexamethasone and insulin to the medium were essential for maintaining liver functions of the cells. These cells synthesized and secreted various serum proteins into the medium. Gluconeogenesis and glycogenolysis were enhanced by glucagon, and lipogenesis was stimulated by insulin. Many enzymes were also induced by various hormones. These activities were very low in freshly isolated cells, but were restored when the cells were cultured for a few days. Markers of plasma membranes, such as 5'-nucleotidase and insulin receptors, were reduced to half the normal levels on freshly isolated cells, but they were restored to the normal levels during culture of the cells without added hormones. Analysis of the profile of amino acids in the medium showed that freshly isolated cells were in a catabolic state of protein turnover and released branched chain amino acids into the medium, but that cultured cells consumed amino acids, not only for protein synthesis, but also for other metabolic processes, such as gluconeogenesis. These findings show that freshly isolated cells have impaired functions and are unsuitable for use in studies of liver metabolism, but that after culture for a few days the cells regain the activity of normal liver and hance become useful for studies of liver functions. Studies with these cells are simpler and give clear results than studies in vivo.
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PMID:Biochemical functions of adult rat hepatocytes in primary culture. 693 75

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

Viable and homogeneous endothelial cells were obtained from isolated guinea pig hearts by application of a special perfusion technique of the coronary system with an isotonic collagenase-trypsin solution and subsequent purification of the dissociated cells by Percoll density gradient centrifugation. The coronary endothelial cells were grown in tissue culture for periods up to 7 months. Serial passage proved to be possible. During logarithmic growth, generation time was found to be 18 h; it could be reduced to 16 h by addition of thrombin to the culture medium. Light, phase contrast and scanning electron microscopy as well as autoradiography revealed that cultured coronary endothelial cells grew as strict monolayers of closely apposed, polygonal large cells. By scanning electron microscopy, it could be demonstrated that the morphology of the cultured cells changes characteristically during attachment of the cells to their substratum. The changes observed were very similar to those of proliferating endothelial cells of isolated coronary vessels kept in organ culture. According to transmission electron microscopy studies, cultured coronary endothelial cells proved to contain only an extremely small number of Weibel-Palade bodies. Nucleoside phosphorylase (EC 2.4.2.5.) and 5'-nucleotidase (EC 3.1.3.5.) were identified in freshly isolated as well as in cultured endothelial cells. Their specific and total activities proved to be much higher than in myocardial tissue, thus indicating a prominent role of nucleotide metabolism in the coronary endothelium.
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PMID:Isolation, identification, and continuous culture of coronary endothelial cells from guinea pig hearts. 728 45

Cytochemical differentiation between blood and lymphatic endothelium has been studied only in microvessels; 5'-nucleotidase (5'Nase) has been reported to be specific for lymphatic and alkaline phosphatase (ALPase) for blood endothelium. Adenylate and guanylate cyclase (AC and GC) have recently been proposed as lymphatic endothelial markers, but conflicting data exist. This study was designed to verify the presence of these enzymes in the endothelium of large vessels and to determine whether they are retained in endothelial cells (ECs) in culture. Segments of bovine mesenteric arteries, veins, and lymphatic collectors, and EC cultures obtained by collagenase treatment of the same vessels, were assayed for 5'Nase, ALPase, AC, and GC, and were observed by transmission electron microscopy. We found ALPase activity in blood and lymphatic vessels, and this was the only enzyme activity consistently retained under culture conditions. 5'Nase was found in lymphatic but not in blood endothelium, as previously reported for microvessels. AC and GC activity was found in blood but not in lymphatic endothelium. Hence, ALPase is not a useful marker to differentiate blood from lymphatic endothelium in large vessels, whereas 5'Nase is specific for lymphatic and AC and GC for blood endothelium. It is not clear why these enzyme activities are not expressed in culture.
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PMID:Cytochemical differentiation between blood and lymphatic endothelium: bovine blood and lymphatic large vessels and endothelial cells in culture. 791 6

Differentiated mammalian cell lines can be established by introducing viral oncogenes into primary cells. Such lines can retain their original specialised functions while being adapted to prolonged life in culture; but most transformed cell lines obtained in this way characteristically show altered properties compared with the primary cells. The result of these changes is that transformed cell lines no longer provide a good model of the original tissue, and indeed often resemble other transformed lines more than the initial cell type. In our laboratory three murine peritoneal macrophage-like cell lines have been isolated by transforming primary cells with SV40 origin-deleted DNA. These lines have been in continuous culture for approximately 1 year and have been shown to express many macrophage-specific properties throughout this time, including Fc receptors and staining for non-specific esterase. The cell lines phagocytosed IgG-coated particles, they were positive for the murine macrophage-specific marker F4/80 and they showed antigen-presentation function. Lysozyme, acid phosphatase, plasminogen activator, collagenase, prostaglandin E2 and 5'-nucleotidase activities have also been detected in these lines. In this paper the method of DNA transformation will be described as well as some of the assays used for the characterization of the three immortalized cell lines.
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PMID:Establishment and characterization of murine macrophage-like cell lines following transformation with simian virus 40 DNA deleted at the origin of replication. 808 36

Interferon-alpha 2b (IFN-alpha 2b) can exert antiproliferative activity in both normal and malignant liver tissue. To study mechanisms of its antiproliferative action, the activity of the enzymes of adenosine metabolism were investigated. We studied 5'-nucleotidase (an adenosine-producing enzyme) and adenosine deaminase (involved in adenosine degradation). Female Wistar rats (3 weeks old) were treated with IFN-alpha 2b for 48 h, as were adult rats (3 months old) and adult rats subjected to partial hepatectomy. During IFN-alpha 2b administration, the activity of 5'-nucleotidase increased in the liver of 3-week-old rats, proportionately more than in adult rats, but the greatest increase was seen in partially hepatectomised rats. The activity of adenosine deaminase decreased in the liver of 3-week-old rats, did not change significantly in 3-month-old rats, but was significantly lower in partially hepatectomised rats. As high adenosine concentrations are toxic for mammalian cells, especially during proliferation, the progressive increase of adenosine production, together with the progressive decrease of its degradation, could be one of the mechanisms of IFN-alpha 2b-induced antiproliferative activity. In vitro studies were performed using collagenase-isolated hepatocytes. They were exposed to IFN-alpha 2b, a cAMP analogue, or both. The incubation of hepatocytes with IFN-alpha 2b did not significantly change the activity of both enzymes, whereas incubation with the cAMP analogue decreased 5'-nucleotidase activity and increased adenosine deaminase activity. The mechanism of IFN-alpha 2b-induced alteration in adenosine metabolism is therefore unclear.
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PMID:Different responses of rat liver adenosine metabolizing enzymes during in vivo and in vitro treatment with interferon-alpha 2b. 979 20

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5'-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5'nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5'-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.
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PMID:Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis. 2486 46


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