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 have developed a new ATP assay for hepatocytes which is simple, rapid and highly sensitive. ATP determination using luciferin-luciferase was performed on hepatocytes separated by perfusion of the liver with collagenase. There was a close correlation between the ATP content of hepatocytes and viable cell numbers. The ATP content of dead cells which were determined by trypan blue dye exclusion test had less than one per cent of levels of viable cells. ATP contents of isolated hepatocytes in Minimum Essential Medium was 6.8 +/- 0.6 x 10(-15) mol/cell at 2 hours after excision of the liver and showed no significant difference compared with that determined at 6 hours. This method was performed to evaluate changes in the cellular ATP content of hepatocytes after partial hepatectomy in rats and transcatheter portal embolization (TPE) in dogs. The ATP content in rat hepatocytes showed a remarkable increase after hepatectomy, with a peak value of 19.1 +/- 1.7 x 10(-15) mol/cell at 24 hours post-surgery. On the other hand, marked atrophy in the embolized lobes and compensatory hypertrophy in the non-embolized lobes were found following TPE in dogs. Cellular ATP content in the non-embolized lobes showed its highest level of 8.7 +/- 2.9 x 10(-15) mol/cell on the third day after TPE, but in the embolized lobes decreased immediately after TPE with significant differences compared with the non-embolized lobes (p < 0.05). Our method may also be applicable to the evaluation of other adenosine phosphate by use of converting enzymes for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A new method for determining cellular ATP content in hepatocytes. 818 8

Cellular respiratory rates are normally determined by metabolic activity, but become rate limited by O2 availability if the cell O2 tension (PO2) falls below a critical value (typically 1-10 Torr). An ability to reduce metabolic activity and energy demand in response to a falling O2 availability might confer an increased resistance to a diminished O2 supply. Isolated rat hepatocytes were studied in primary culture under controlled O2 tensions. Cells were obtained by collagenase digestion and seeded into nutritive media in control and experimental spinner flasks at identical cell densities. Cells subjected to rapid reduction in PO2 (100-->0 Torr over < 40 min) exhibited undiminished O2 uptake until PO2 fell below 10 Torr. By contrast, when cell PO2 was reduced over several hours, significant decreases in O2 uptake became evident at O2 tensions as high as 70 Torr. These decreases were associated with a reduction in ATP concentration and an increase in NAD(P)H, compared with rapidly deoxygenated cells at the same PO2. No loss in cell viability was detected after 24 h at reduced PO2. The decrease in respiratory rate was associated with an increased rate of lactic acid production relative to normoxic controls. Restoration of normoxia was associated with an immediate return of O2 uptake to control levels. These results demonstrate that hepatocytes are capable of reversibly decreasing metabolic activity and O2 demand during sustained moderate reductions in PO2, via a mechanism that appears to involve an inhibition of mitochondrial function other than O2 supply limitation. This response may alter cellular susceptibility to physiological stresses including hypoxia.
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PMID:Oxygen conformance of cellular respiration in hepatocytes. 823 74

1. In rat whole portal veins, guanabenz (100 nM to 10 microM) and antazoline (100 nM to 100 microM) each increased the amplitude, frequency and duration of spontaneous contractions. In addition, guanabenz (30 microM) and antazoline (30 microM) each antagonized the ability of levcromakalim (3 nM to 10 microM) to inhibit the spontaneous contractions of this tissue. 2. Whole-cell voltage-clamp recordings were made from freshly-isolated rat portal vein cells dispersed by a collagenase/pronase enzyme treatment. The ability of several agents (antazoline, cirazoline, clonidine, guanabenz and phentolamine, each containing an imidazoline or guanidine moiety), to modulate potassium (K) currents and to inhibit the actions of levcromakalim was investigated. 3. Antazoline, cirazoline, clonidine, guanabenz and phentolamine (each at a concentration of 30 microM) had little effect on control non-inactivating currents but inhibited the delayed-rectifier current, IK(V). 4. Levcromakalim (1 microM) induced a non-inactivating current, IK(ATP), and also inhibited the delayed rectifier current, IK(V). 5. Glibenclamide (1 microM) had no effect on control delayed rectifier or non-inactivating currents, but it inhibited the simultaneous induction of IK(ATP) and reduction of IK(V) produced by levcromakalim (1 microM). 6. Antazoline, cirazoline, clonidine and guanabenz (each at a concentration of 30 microM) prevented the induction of IK(ATP) by levcromakalim (1 microM). Phentolamine (30 microM) and clonidine (30 microM) each inhibited the IK(ATP) generated by levcromakalim (1 microM). 7. It is concluded that a variety of agents which possess either an imidazoline (antazoline, cirazoline, clonidine and phentolamine) or a guanidine (guanabenz) moiety within their structure inhibit the delayed rectifier current, IK(V). This action may thus be mediated via a so-called non-adrenoceptor imidazoline binding site. Furthermore, the ability of these ligands to inhibit IK(V) and to antagonize both the induction of IK(ATP) and the vasorelaxation produced by levcromakalim is consistent with the view that the channel (KATP) which underlies IK(ATP) is a voltage-insensitive state of the delayed rectifier K-channel (Kv).
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PMID:Antagonism of levcromakalim by imidazoline- and guanidine-derivatives in rat portal vein: involvement of the delayed rectifier. 830 1

This study aimed to develop methods for the large-scale preparation of hepatocytes from large animal livers and for the mass cryopreservation of isolated hepatocytes. Isolated hepatocytes were obtained from Beagle dogs weighing around 10 kg by collagenase digestion plus preperfusion with the Ca2+ chelator ethylene glycol bis N,N'-tetraacetic acid. The cell yield was 2.1 +/- 0.45 x 10(10)/liver with 90% viability by the trypan blue dye exclusion test, and the estimated cell yield was 72 +/- 13%. The optimal conditions were large-scale cryopreservation of dog hepatocytes were (i) a freezing rate of 1-10 degrees C/min, (ii) a dimethyl sulfoxide (Me2SO) concentration of 20% (final concentration: 10%), (iii) a cell density that did not exceed 10(8)/ml, and (iv) rapid thawing in a 37 degrees C water bath. The viability of preserved hepatocytes was 75 +/- 3.0% and the estimated recovery rate was approximately 50%. Preserved hepatocytes showed 20-50% of the metabolic activity of fresh cells, as assessed by ammonia and fructose loading tests, ATP content, and [14C]leucine uptake. Following culture after thawing, the morphological normalization of preserved cells was confirmed by light and electron microscopy.
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PMID:Large-scale cryopreservation of isolated dog hepatocytes. 844 Jan 24

In isolated mitochondria, t-butylhydroperoxide (t-BuOOH) and other pro-oxidants cause a permeability transition characterized by increased permeability to small ions, swelling and loss of membrane potential. Cyclosporin A and trifluoperazine inhibit this permeability transition. Here, we investigated the role of the mitochondrial permeability transition in lethal cellular injury from t-BuOOH. Hepatocytes from fasted rats were isolated by collagenase perfusion, and cell viability was assessed by propidium iodide fluorescence. t-BuOOH caused dose- and time-dependent cell killing. Fructose, a substrate for glycolytic ATP formation, protected at lower (< or = 100 microM), but not at higher concentrations of t-BuOOH. In fructose-treated cells, oligomycin (10 micrograms/ml) delayed cell killing after 100 to 300 microM t-BuOOH, whereas cyclosporin A (0.5 microM) plus trifluoperazine (5 microM) even more potently reduced lethal injury. In hepatocyte suspensions, 100 microM t-BuOOH caused mitochondrial depolarization as determined by release of rhodamine 123. Cyclosporin A plus trifluoperazine in the presence of fructose substantially reduced release of rhodamine 123. Similarly, in single cultured hepatocytes viewed by laser scanning confocal microscopy, t-BuOOH caused leakage of rhodamine 123 from mitochondria, an event which preceded cell death and which was delayed by fructose in combination with cyclosporin A plus trifluoperazine. At 1 mM, t-BuOOH inhibited glycolysis, and fructose in combination with either oligomycin or cyclosporin A plus trifluoperazine had only a short-lived protective effect. In conclusion, t-BuOOH toxicity was progressive with increasing dosages. At low t-BuOOH (< or = 50 microM), mitochondrial ATP synthetic capacity was inhibited, but not uncoupled.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine. 847 21

We have prepared purified cytotrophoblasts from human term placentas and examined the sensitivity of fura-2 loaded cells to the nucleotides ATP and UTP and to changes in extracellular Ca2+ concentration ([Ca2+]o). Purified cytotrophoblasts were obtained by collagenase digestion and separation according to density using self-generated Percoll gradients. The cytotrophoblast fraction was free of red cell and largely free of white cell contamination (as assessed by uniformly negative staining for vimentin and the failure of > 90% of fura-2 loaded cells to respond to the chemotactic peptide fMet-Leu-Phe). Purified cells secreted progesterone in a linear fashion over several hours in the presence of 25-hydroxycholesterol. The cells ranged in size from approximately 7.5 to 50 microns in diameter as described previously for purified cytotrophoblasts, and an analysis of cells for sensitivity to [Ca2+]o or nucleotides suggested functional heterogeneity within the cytotrophoblast population. Small cells (7.5-10 microns) were negative for cytokeratin-8 and, after loading with fura-2, were insensitive to extracellular nucleotides but sensitive to elevations in [Ca2+]o. Medium-sized cells (12-20 microns) were largely cytokeratin-positive (70% of cells) and sensitive to both ATP and UTP but largely insensitive to [Ca2+]o. Large cells (25-50 microns) were uniformly cytokeratin-positive (100% of cells) and, after fura-2 loading, sensitive to both [Ca2+]o and extracellular ATP or UTP. We examined the likely origin of small, medium and large cytotrophoblasts using an immunomagnetic cell sorting procedure that separates villous cytotrophoblasts (which do not express major histocompatibility class I antigens) from extravillous cytotrophoblasts. This procedure resulted in the selective sedimentation of almost all medium and large cells, leading to the conclusion that the small cells were villous cytotrophoblasts whereas medium and large cells were predominantly extravillous in origin. The data suggest that small, medium and large cytotrophoblasts have distinct roles in the function of the term placenta.
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PMID:Functional heterogeneity of human term cytotrophoblasts revealed by differential sensitivity to extracellular Ca2+ and nucleotides. 867 46

We describe a method to isolate epithelial cells from gallbladders of Necturus maculosus with preserved structural and functional polarity. Isolation was carried out with a mixture of collagenase and protease, with only a brief exposure to a divalent-cation-free medium. About 40% of the isolated epithelial cells had a "figure-eight" shape and retained metabolic and cell membrane integrity. Figure-eight cells display features consistent with preserved polarity for several hours, including the following: 1) the "apical" and "basolateral" membrane domains were differentially labeled by a hydrophobic fluorescent dye; 2) freeze fracture electron microscopy verified two plasma membrane domains differing in the presence of microvilli and folds and separated by tight junctions; 3) proteins such as ZO-1, NHE3, and Na(+)-K(+)-ATPase remained localized in the junctional, apical, and basolateral regions, respectively; 4) after apical surface exposure to wheat germ agglutinin, the label remained in the apical membrane after cell isolation; and 5) patch-clamp experiments demonstrated polarized expression of K+ channels. Polarity was rapidly lost after removal of extracellular Ca2+, exposure to trypsin, or ATP depletion. Therefore, this preparation allows for structural and functional studies of epithelial transport in single cells retaining the essential features present in the assembled epithelium.
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PMID:Preservation of structural and functional polarity in isolated epithelial cells. 876 71

Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, i.p.) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 microns) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments ( < 4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.
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PMID:Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. 880 57

Prolonged bleeding by the host after the leech ceases to feed and several reports that the use of leeches restores blood flow in the microcirculation after plastic surgery led us to search for inhibitors of platelet aggregation in Hirudo medicinalis saliva. Dilute leech saliva was collected by phagostimulating starved leeches with a solution of arginine in saline. The saliva is shown to inhibit human platelet aggregation induced by thrombin, collagen, adenosine diphosphate (ADP), epinephrine, platelet activating factor (1-O-alkyl-2-acetyl-sn-3-glycerophophoryl choline [PAF]), and arachidonic acid. We have isolated the PAF inhibitor and found it to be an amphipathic phosphoglyceride. We have also purified apyrase adenosine triphosphate ([ATP] diphosphohydrolase), which inhibits ADP-induced platelet aggregation, and have described collagenase. Besides well-known hirudin, Hirudo saliva contains a potent inhibitor of coagulation factor Xa. We also report antiaggregant and anticoagulant activities in the crop content of the closely related Nile leech, Limnatis nilotica. Anticoagulants of hematophagous species are surveyed. We have used medicinal leeches in plastic surgery for decompression of skin flaps and in patients with postphlebitic syndrome and peripheral arterial occlusions. Preliminary results indicate certain beneficial effects of leech therapy.
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PMID:Platelet aggregation and coagulation inhibitors in leech saliva and their roles in leech therapy. 883 13

ATP-sensitive K+ channels (KATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [3H]-P1075 ([3H]-N-cyano-N'-(1, 1-dimethylpropyl)-N"-3-pyridylguanidine), a selective opener of KATP channels, in rat glomerular preparations. Equilibrium (saturation, competition) and kinetic experiments indicated that [3H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg-1 glomerular protein. The association rate constant of the complex was 6,5 x 10(7) M-1 min-1; dissociation occurred with a half-time of 6.2 min. Specific [3H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [3H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [3H]-P1075 binding was inhibited by representatives of the different classes of KATP channel openers and by sulphonylurea-type blockers with inhibition constants similar to those obtained in rat aortic rings. It is concluded that rat glomerular preparations possess specific binding sites for KATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 microns-2) is much higher than that of KATP channels in smooth muscle.
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PMID:Binding of [3H]-P1075, an opener of ATP-sensitive K+ channels, to rat glomerular preparations. 889 48


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