Characterization of Isolated Rat‐Liver Cells Made Permeable with Filipin

Gankema, Henk; Laanen, Elly A.; Groen, Albert K.; Tager, J. M.

doi:10.1111/j.1432-1033.1981.tb05623.x

Cited by 29 publications

(16 citation statements)

References 46 publications

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“…The results in Table 1 confirm previous reports that filipin-treated hepatocytes provide a suitable model for studying enzymes and organelles in their macromolecular environment in situ (Jorgensen & Nordlie, 1980; Gankema et al, 1981). However, before meaningful conclusions could be drawn from the results, it was necessary to investigate the time of onset of the filipin response and the effect of prolonged incubation periods on the integrity of both the cell and the mitochondrial membrane.…”

Section: Effect Of Time Of Incubation On Pyruvate Carboxylation Infilsupporting

confidence: 84%

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes

Allan¹,

Chisholm²,

Titheradge³

1983

Biochemical Journal

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A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure.

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Section: Effect Of Time Of Incubation On Pyruvate Carboxylation Infilsupporting

confidence: 84%

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes

Allan¹,

Chisholm²,

Titheradge³

1983

Biochemical Journal

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“…Attempts of Krebs et al [3] to obtain gluconeogenesis in liver homogenates from rats and several other species were not successful and this system as well as the one of Mendicino and Utter [2] has not been used for further studies. Beside these investigations on the whole chain of gluconeogenesis in cellfree systems, some studies on partial sequences in subcellular fractions of rat liver have been made by the group of Lardy [8,9], by McDermott and Veneziale [lo] and by ourselves Recently, liver cells rendered permeable to substrates and effectors by treatment with filipin were proposed as a possible [I 1,121. model for studying metabolic regulation by low molecular weight compounds [13]. A different approach to the same problem is described in the present study which demonstrates the conversion of malate to glucose 6-phosphate at rates comparable to those observed in rat hepatocytes in a cell-free system derived from rat liver.…”

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confidence: 71%

Gluconeogenesis in vitro

Mörikofer‐Zwez

Stoecklin

Walter

1982

European Journal of Biochemistry

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A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors at near physiological concentrations was shown to form glucose 6-phosphate from malate + 3-phosphoglycerate at a rate of 1.11 0.09 pmol . min-' . g liver-' (mean f SEM, n = 9, 30°C). At least 70% of glucose 6-phosphate formed was derived from malate as calculated from experiments with [U-14C]malate. The indicated rates were measured between 10 min and 30 min incubation time when the system was near steady state with respect to the lactate/pyruvate ratio and to most of the gluconeogenic intermediates.In the absence of mitochondria, the rate of formation of glucose 6-phosphate from malate was about seven times lower than in their presence. A comparison between incubations carried out in presence or absence of mitochondria revealed that mitochondria decreased the lactate/pyruvate ratio and increased the ratio of (ATP + ITP)/(ADP + IDP). It could be shown that under the present incubation conditions, formation of glucose 6-phosphate was closely linked to the ratio of (ATP + ITP)/(ADP + IDP) whereas changing redox ratios had little influence on the gluconeogenic rate.Regulation of gluconeogenesis in liver is known to involve mitochondrial, cytosolic as well as microsomal reactions and has mainly been studied in liver slices, perfused liver or hepatocytes (for a review see [I]). Two early attempts have, however, been made to set up gluconeogenic systems which are readily accessible to low molecular weight effectors in order to obtain more detailed information on regulatory mechanisms. Mendicino and Utter [2] showed that a recombination of purified gluconeogenic enzymes was able to form glucose 6-phosphate from 3-phosphoglycerate or fumarate in the presence of chicken liver mitochondria. Furthermore, the group of Krebs [3,4] described a cell-free system of pigeon liver homogenate which formed glucose from lactate at physiological rates. In both cases, systems were employed where phosphoenolpyruvate carboxykinase is localized within the mitochondria [S,6] which is not the case in rat liver [7]. Attempts of Krebs et al. [3] to obtain gluconeogenesis in liver homogenates from rats and several other species were not successful and this system as well as the one of Mendicino and Utter [2] has not been used for further studies. Beside these investigations on the whole chain of gluconeogenesis in cellfree systems, some studies on partial sequences in subcellular fractions of rat liver have been made by the group of Lardy [8,9], by McDermott and Veneziale [lo] and by ourselves Recently, liver cells rendered permeable to substrates and effectors by treatment with filipin were proposed as a possible [I 1,121. model for studying metabolic regulation by low molecular weight compounds [13]. A different approach to the same problem is described in the present study which demonstrates the conversion of malate to glucose 6-phosphate at rates comparable to those observed in rat hepatocytes in a cell-free system derive...

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“…The procedures for treatments with filipin described by Jorgenson and Nordlie (6) and Gankema et al (7) were followed as closely as possible.…”

Section: Preparation Of Permeabilized Ceilsmentioning

confidence: 99%

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

McEwen¹,

Arion²

1985

The Journal of Cell Biology

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Pathogenic staphylococci secrete a number of exotoxins, including a-toxin, aToxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used a-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage <_22%. Treatment conditions included incubation for 5-10 min at 37°C and pH 7.0 with an a-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml a-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, a-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.The study of the relationship of enzymic activities to internal cell structure requires the development of assay systems that can determine these activities in situ. For most reactions, the development of such assays requires circumventing the permeability barrier presented by the plasma membrane. The use of toluene (1, 2) or detergents to render the plasma membrane permeable to ionic substrates is unsatisfactory since such agents induce permeability changes in intracellular membranes. Toluene treatment also results in large morphological changes in the internal cell structure (2). The ideal reagent would be one that selectively reacts with the plasma membrane, does not induce any alterations of cellular morphology, and does not cause leakage of cytosolic enzymes.Filipin (3-7), digitonin (8-12), and saponin (13-15), all of which complex with membrane-bound cholesterol, have been used to create permeability in cells to a variety of substances. Since the plasma membrane has a higher cholesterol content than most internal membranes (e.g., mitochondria and the 1922 endoplasmic reticulum [ER]'), it has been postulated that these agents would react preferentially with the plasma membrane (6, 7, 1 l, 13). In spite of this selective interaction, the use of filipin or digitonin can cause extensive changes in cell morphology (12,16,17). 2 The rough ER is especially vulnerable, and other studies show that these agents alter the kinetics of ER enzymes (Lange, A. J., B. F. McEwen, and W. J. Arion, unpublished observations; and references 18-20), which suggests the agents interact with the ER membrane or E...

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Characterization of Isolated Rat‐Liver Cells Made Permeable with Filipin

Cited by 29 publications

References 46 publications

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes

Gluconeogenesis in vitro

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

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