共查询到20条相似文献,搜索用时 15 毫秒
1.
Mark A. Horrum Richard B. Jennett Robert E. Ecklund Richard B. Tobin 《Molecular and cellular biochemistry》1986,71(1):79-85
Summary The effects of the microtubular inhibitor, podophyllotoxin, on mitochondrial respiration were determined using isolated, digitonin-permeabilized hepatocytes and isolated mitochondria. In hepatocytes, podophyllotoxin (1.5 mM) inhibited coupled and uncoupled respiration of both FAD and NAD-linked substrates. In mitochondria, podophyllotoxin inhibited State III respiration, prevented the return to State IV respiration, and inhibited uncoupled respiration. There was no inhibition of ascorbate/TMPD oxidation in either the hepatocytes or the mitochondria. Podophyllotoxin had no effect upon oligomycin inhibition of coupled respiration. Oligomycin had no effect on the podophyllotoxin-inhibition of uncoupled respiration in either hepatocytes or mitochondria. The results indicate that podophyllotoxin alters electron flow at a site early in the electron transport chain. 相似文献
2.
Morphometric and biochemical techniques were used to analyze hepatic glycogen, endoplasmic reticulum, and mitochondrial matrix granules in rats treated with compound 48/80 to induce an anaphylactic-like state of shock. Thirty minutes after insult there was a significant decrease in glycogen and mitochondrial matrix granules, an increase in rough endoplasmic reticulum (RER), and no change in smooth endoplasmic reticulum (SER). Less glycogen in experimental rats substantiated a previously described glycogenolytic response to compound 48/80. The decrease in matrix granules implies a loss and/or shift in intramitochondrial calcium as occurs in epinephrine-induced glycogenolysis in the rat. Since other glycogenolytic agents, e.g. glucagon, and starvation stimulate an increase in SER presumably from RER, the present morphological data suggest the increase in RER may precede proliferation of SER from RER. 相似文献
3.
Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with -amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.Abbreviations SER
smooth endoplasmic reticulum
- RER
rough endoplasmic reticulum
- PMS
post mitochondrial supernatant
- MES
2-(N-morpholino) ethane sulfonic acid
- PMSF
phenylmethylsulfonyl fluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
4.
Charles A. Tyson Carol E. Green Susanna E. LeValley Robert J. Stephens 《In vitro cellular & developmental biology. Plant》1982,18(11):945-951
Summary Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique
and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times
higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin.
A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial
(5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant
changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar
in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these
preparations as a model for Fe mobilization from Fe-loaded animals.
This work is supported by National Institutes of Health Grants AM 25647-03 (M. Dawson, Principal Investigator) and GM 28158-01
(C. Tyson, Principal Investigator). The technical assistance of Mr. Jack E. Dabbs, Mr. Charles Hart, and Mr. Randy Douglas
is acknowledged. 相似文献
5.
A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ 总被引:1,自引:0,他引:1
We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites. 相似文献
6.
M. Ozturk F. Lemonnier D. Cresteil J. Scotto A. Lemonnier 《Chemico-biological interactions》1984,51(1):63-76
The biochemical and morphological effects of 2, 10 and 100 mM of D-galactosamine (GalN) were studied in isolated rat hepatocytes during 2 h of incubation. Lactate dehydrogenase (LDH), alanine aminotransferase (ALAT) and cell viability did not change, whatever the concentration used. The variations observed, which were dose dependent, included a large drop in ATP levels and inhibition of RNA and protein synthesis. A very high concentration of GalN was necessary, however, to induce a significant decline in methionine adenosyltransferase activity compared to control cells.The use of L-[methyl-14C]methionine during cell incubation with GalN demonstrated a decrease of S-adenosyl-L-methionine (SAMe) and an accumulation of L-methionine content related to the GalN concentration. These results suggested that an hepatotoxic agent such as GalN was able to induce disturbances of methionine metabolism.Some of the ultrastructural changes observed were different from those previously found in vivo, in rats given GalN intraperitoneally, underlining the marked difference between in vivo and in vitro intoxication. 相似文献
7.
Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes
James L. Driscoll Nancy T. Hayner Rhonda Williams-Holland Geraldine Spies-Karotkin Pierre M. Galletti Hugo O. Jauregui 《In vitro cellular & developmental biology. Plant》1982,18(10):835-842
Summary The sulfonic acid dye, phenolsulfonphthalein (PSP or phenol red), has been incorporated as a pH indicator in many tissue culture
media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway,
time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic
studies coupled with β-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About
20% of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h,
and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect
of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations
of less than 100μM there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially
metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cells). Therefore, its incorporation
into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s)
of glucuronidation or metabolic pathways thought to be affected by this anionic dye.
This study was supported in part by NIH Grants HL-11945-11 and 1 R01 AM 26520-01A1. 相似文献
8.
Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretioninTcells.However,whetherhomocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivoand in vitrostudies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition ofERstress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming. 相似文献
9.
Dongmei Cheng Allison Weckerle Yi Yu Lijun Ma Xuewei Zhu Mariana Murea Barry I. Freedman John S. Parks Gregory S. Shelness 《Journal of lipid research》2015,56(8):1583-1593
Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaperones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver. 相似文献
10.
A new, improved procedure for isolating mitochondria from ascites tumor cells is described. The unique feature of this technique is the use of digitonin to make the cells susceptible to disruption by Teflon pestle/glass vessel homogenization. The yield and respiratory control ratios of mitochondria isolated by this method from murine Ehrlich ascites tumor cells and rat AS30-D ascites hepatoma cells are significantly better than those obtained for mitochondria isolated by the commonly employed Nagarse method, which involves the use of proteolytic enzymes. Moreover, mitochondria isolated by this new procedure from three different lines of tumors exhibit respiratory control ratios with both adenosine diphosphate and a respiratory uncoupler comparable to those obtained with mitochondria present in situ within digitonin-permeabilized tumor cells. 相似文献
11.
Prof. K. Smetana R. L. Ochs R. K. Busch L. C. Yeoman H. Busch 《Cell and tissue research》1987,249(1):235-239
Summary The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23. 相似文献
12.
Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains 总被引:8,自引:0,他引:8
Kazunori Furukawa Tomiko Shimada Patricia England Yohichi Mochizuki Gary M. Williams 《In vitro cellular & developmental biology. Plant》1987,23(5):339-348
Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible
establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats
by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells
different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded
numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority
of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation
at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in
the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity,
whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics
of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial
cells propagable in culture were derived from a cell type other than the hepatocyte. 相似文献
13.
Inositol (1,4,5)trisphosphate-promoted Ca2+ release from microsomal fractions of rat liver 总被引:23,自引:0,他引:23
Crude mitochondrial fractions containing a substantial amount of microsomes accumulate Ca2+ in the presence of ATP, ruthenium red and oligomycin. A proportion of this accumulated Ca2+ is released by the addition of low concentrations (ca. 1 microM) of inositol (1,4,5) trisphosphate . Under some conditions the release is transient, and evidence is presented which suggests that this is due to inhomogeneity in the vesicle population. (1,4,5)inositol trisphosphate -induced Ca2+ release can also be demonstrated, under appropriate experimental conditions, in a more purified microsomal fraction essentially free of mitochondria. 相似文献
14.
Formation of cylindrical multicellular aggregate (cylindroid) and expression of liver specific functions of primary rat hepatocytes 总被引:2,自引:0,他引:2
Hiroshi Mizumoto Masashi Hayakami Kohji Nakazawa Hiroyuki Ijima Kazumori Funatsu 《Cytotechnology》1999,31(1-2):69-75
In our studies of the development of a hybrid artificial liver, we investigated the formation of cylindrical multicellular
aggregate (cylindroid) of primary rat hepatocytes on a pressed sheet of polyurethane foam (pressed–PUF) as a culture substratum.
Hepatocytes formed cylindroids by attaching to a pressed–PUF surface, peeling off from the surface and aggregating. The diameter
and length of most cylindroids were approximately 200–500 μm and 500 μm–2 mm, respectively. The activities of liver specific
functions (albumin secretion and ammonia metabolism) of hepatocyte cylindroids were equivalent to or higher than those of
hepatocyte spheroids. These results suggest that hepatocyte cylindroids can maintain highly differentiated functions longer
than hepatocyte spheroids, and that a PUF/cylindroid culture may be effective to develop of a hybrid artificial liver.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
《Molecular membrane biology》2013,30(3):283-288
The entry of substrates into, and the export of glururonides from, the lumen of hepatic endoplasmic reticulum (ER) in vitro (sealed microsomes) has been measured using radioactivity-labelled materials and a rapid filtration assay. Analysis of liver microsomes from a jaundiced patient showed the accumulation of bilirubin glucuronides within the lumen of the ER. Further analysis of these hepatic microsomes revealed that newly synthesized 1-naphthol glucuronide could exit from the microsomes whereas billrubin glucuronide was accumulated within the microsomes. These results suggest the existence of mechanisms for the sorting of small molecules, destined for export through bile canalicular or basolateral plasma membranes, by ER. Furthermore, these sorting processes may be regulated by specific transporters within the ER. 相似文献
16.
Gert Kreibich Maria Czak-Graham Ruth Grebenau Winnie Mok Enrique Rodriguez-Boulan David D. Sabatini 《Journal of cellular biochemistry》1978,8(3):279-302
Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: (i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; (ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detegent Kyro EOB; (iii) in intact rough microsomes ribophorins can be crosslinked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and “rough-inverted” vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents sugest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them. 相似文献
17.
G. Bruscalupi L. Conti Devirgiliis S. Leoni F. Piemonte A. Trentalance 《Bioscience reports》1986,6(8):735-740
Acyl CoA: cholesterol acyl transferase (ACAT) activity presents marked oscillations and differential sensitivity to the in vitro stimulation of the kinase-phosphatase modulatory system in the perinatal rat liver.The regulation of this enzyme activity by some modulators generally active in adulthood, such as cholesterol, lipoproteins and mevalonate, has been studied in hepatocytes isolated at different developmental stages. A lack of effect of mevalonate and a positive effort of lipoprotein cholesterol have been observed at the fetal and neonatal stages.A differential prevalence is suggested of one of the two modulatory mechanisms (phosphorylation-dephosphorylation system, or substrate effect) at each developmental stage. 相似文献
18.
Ubaldo Armato Paola G. Andreis Enrica Draghi Loreta Mengato 《In vitro cellular & developmental biology. Plant》1978,14(6):479-484
Summary Studies employing [3H]thymidine and radioautography as well as colchicine and Feulgen staining of DNA showed that up to 19-fold increases in the
degree of cell crowding in vitro, i.e. from 1.45 to 27.55×104 cells per specimen, did not change the rates of entry into DNA synthesis and mitosis of cultivated primary neonatal rat hepatocytes. 相似文献
19.
Liang Qiao Geoffrey C. Farrell 《In vitro cellular & developmental biology. Animal》1999,35(7):417-424
Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high
and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were
greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei
in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures
but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on
collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis.
The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells
were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen
1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures,
aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures
of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated,
at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent
manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell
density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes. 相似文献
20.
To improve long-term expression of drug biotransformation activities in hepatocytes, we have examined the suitability of several epithelial-like cell lines (MDCK, MS and L-132) for supporting functional co-cultures with rat hepatocytes. Cells were selected on the basis of their compatibility with hepatocytes, formation of stable monolayers in the absence of serum and lack of drug biotransformation activities. The expression of individual elements of the biotransformation system was evaluated in these co-cultures. Co-cultured hepatocytes remained viable and showed a characteristic polygonal shape for more than a week. Depending on the cell line used, levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities of co-cultured hepatocytes oscillated between 24–47% of their initial value after 4 days in culture. The highest levels of monooxygenase activity were found in hepatocytes co-cultured with MS cells (41–47%). In contrast, these activities decreased to 6% when hepatocytes were maintained in pure culture for the same period. The activities of the conjugating enzymes UDP-glucuronyltransferase and glutathione S-transferase were maintained at nearly the initial levels during the complete period of study, both in pure and mixed-cultures, regardless of the cell line used. MS cells adapted themselves much better to serum-free culture conditions, and the co-culture with rat hepatocyte was technically easier. After one week, total cytochrome P450 and reduced glutathione in rat hepatocytes/MS co-cultures were 31% and 127% respectively of the day O values, whereas they were undetectable in pure culture. A clear induction of monooxygenase activities by methylcholanthrene, phenobarbital and ethanol could be observed by the 5th day in MS cells/hepatocyte co-cultures. The fact that the results of our work show the suitability of MS cells, an epithelial-derived cell line, for improving the expression of biotransformation enzymes of cultured hepatocytes opens new possibilities of simplifying co-cultures for their use in drug-metabolism studies.Abbreviations AHH
aryl hydrocarbon hydroxylase
- CDNB
1-chloro-2,4-dinitrobenzene
- DMEM
Dulbecco's modified Eagle's medium
- ECOD
7-ethoxycoumarin O-deethylase
- EDTA
ethylenediamine tetraacetic acid
- Et-OH
ethanol
- GSH
reduced glutathione
- GSH-t
glutathione S-transferase
- MC
3-methylcholanthrene
- PB
phenobarbital
- UDP-Gt
UDP-glucuronyltransferase 相似文献