Analysis by flow cytometry of rat hepatocytes from different acinar zones

Vargas, Jose Luis; O’Connor, Enrique; Roche, Enrique; Knecht, Erwin; Grisolı́a, Santiago

doi:10.1016/0006-291x(87)90964-8

Cited by 18 publications

(10 citation statements)

References 15 publications

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“…Because of both their structural properties and physiological commitment, as well as their metabolic sensitivity to the incubation conditions, isolated rat hepatocytes were considered ideal for this study (1,4,12,19,35). Our results show that, once the appropriate dye-to-cell ratio is established, the flow cytometric kinetic assay of Rh123 uptake at low concentrations is a fast and specific measurement of MMP and can be applied conveniently to detect metabolic and toxic effects on the mitochondria1 compartment in liver cells, as well as to assess the metabolic state of isolated cells along extended incubations.…”

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confidence: 99%

A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry

et al. 1994

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Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 pg/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for certain applications in which labile cells are used or which demand rapid or repeated analysis. In this paper we describe a rapid kinetic assay of MMP in isolated rat hepatocytes, based upon the quantitation of the initial rate of Rh123 uptake by living cells, selected by their scattering properties. The results indicate that at an appropriate dye-to-cell ratio (in our experiments, 50 ng Rh123/ml for 250,000300,000 cells/ml), the initial rate of Rh123 uptake is a highly reproducible and sensitive parameter for estimation of MMP, as demonstrated by the effects of substrates and inhibitors of the glycolytic pathway and mitochondrial respiration. Because of its simplicity, rapidity (about 5 min) and metabolic implications, this assay would be also suitable for the routine evaluation of metabolic state of cell suspensions, as a complementary test to the standard dualstaining tests of viability. Other possible applications in screening pharmacologic and toxicological analysis are discussed. o 1994 Wiley-Liss, Ine.Key terms: Mitochondria, mitochondrial membrane potential, rhodamine 123, flow cytometry, hepatocyte, glucose metabolism Mitochondrial membrane potential (MMP) is a key parameter to assess cellular energy metabolism under physiological and pathological conditions (5). However, the conventional approach of studying isolated mitochondria in vitro does not allow integration of the mitochondrial function into the whole cell.Flow cytometry provides convenient methods for the analysis of MMP in situ. Recently, most flow cytometric studies on mitochondrial function have been performed with rhodamine 123 (Rh123), a lipophilic cationic fluorochrome which is incorporated by mitochondria according to transmembrane potential (6,9,11,14,20,22). Rh123 has been applied extensively in flow cytometric studies of mitochondrial metabolism (2,30,33) and metabolic heterogeneity (341, cell activation (81, differentiation (71, oncogenic transformation (32), stem cell characterization and purification (37,381, aging (16), and apoptotic death (lo), as well as for assessment of cytotoxicity (20,22,23,24) and drug resistance (15,17). Some of the multiple applications of Rh123 have been reviewed by Ronot et al. (26,271 and Chen (5,6).The most usual techniques to study MMP by flow cytometry in whole cells follow the one described by Johnson et al. (13,14), with slight modifications. Following a 30-60 min equilibration of cells with Rh123

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A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry

et al. 1994

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“…Using electron microscopic immunocytochemical procedures we have previously found that there is homogeneity in the content of CPS and GDH among mitochondria from the same hepatocyte (intracellular homogeneity of mitochondria) [20,21]. Here, we have measured the relative distribution of GDH, CPS and OTC in isolated hepatocyte populations [15]. As shown above, the content of these mitochondrial enzymes is different in the three hepatocyte populations studied.…”

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confidence: 99%

“…To assess the intraacinar origin of the populations, we measured: PK, a perivenous hepatocyte marker, and AAT and LDH, periportal hepatocyte markers [11,12]. The activity of AAT and LDH increases from F1 (light hepatocytes) to F3 (heavy hepatocytes) populations, while that of PK decreases [15].…”

Section: Resultsmentioning

confidence: 99%

“…The discontinuous Percoll gradients contained in the interlayers three populations with a similar [15]. To assess the intraacinar origin of the populations, we measured: PK, a perivenous hepatocyte marker, and AAT and LDH, periportal hepatocyte markers [11,12].…”

Section: Resultsmentioning

confidence: 99%

“…Hepatocytes with a viability higher than 95°70 (measured by trypan blue exclusion) were used. Three cell populations were obtained as described in [15].…”

Section: Cell Preparationmentioning

confidence: 99%

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Differences in the half‐lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity

et al. 1987

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The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half-life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half-lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3-times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half-life differences of the enzymes studied.

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The liver and intracellular digestion: How liver cells eat!

Yamazaki

LaRusso

1989

Hepatology

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The liver plays a pivotal role in the turnover of macromolecules because: (i) it is supplied by the nutrientrich portal blood (ii) it has a unique anatomy which permits direct physical contact between sinusoidal blood and liver cells; (iii) it is the principal source of most serum proteins, and (iv) it is composed of several types of cells that contain receptors and other membrane specializations that optimize removal of circulating materials. Major cellular activities that contribute to such macromolecular turnover include synthesis, extraction, metabolism and excretion ( l ) , activities that may be included under the general term "cellular processing." Whereas the physiological importance of the synthetic activities of the liver has long been appreciated (a), hepatic degradative or digestive processes have not received the same attention, even though Schoenheimer (3) emphasized the importance of degradation relative to synthesis in his classic work over four decades ago. Studies in the last 10 years have shown that a heterogeneous array of materials can be processed in liver cells, the cells often releasing resultant degradative products into bile (4). Recently, we reviewed the vacuolar components of intracellular digestion and reintroduced the concept of "cellular gastroenterology" (5, 6), a concept originally suggested by Christian de Duve over 30 years ago when he discovered the lysosome (7-9). Indeed, a quite reasonable series of analogies can be made between the digestive tract of the cell and that of the whole organism, as will be fully discussed later. For these reasons, it seemed timely to summarize current knowledge concerning intracellular digestion in liver cells. Although a number of recent reviews addressing specific mechanisms and regulatory aspects of intracellular digestion are available (10-19), none has been written from the unique perspective of the experimental hepatologist. Thus, the purpose of this overview is not to itemize further the available detailed information on intracellular digestion which has been covered extensively in preof Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905. vious reviews; rather, we intend to update and reorganize information on selected aspects of intracellular digestive processes in relation to experimental and clinical hepatology. We will place particular emphasis on the intracellular degradation of proteins ( i e . proteolysis) by hepatic parenchymal cells, since 99% of cellular proteins in the liver reside in hepatocytes (18,20) and since protein breakdown has been more extensively studied than the intracellular digestion of lipids and carbohydrates. We will also focus on lysosomes (i.e. ubiquitous intracellular organelles containing over 50 hydrolytic enzymes capable of degrading virtually all cellular constituents) because of our ongoing interest in these organelles and because they represent the best-characterized intracellular compartment for proteolysis. It should be added that many of the processes described in this review...

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Analysis by flow cytometry of rat hepatocytes from different acinar zones

Cited by 18 publications

References 15 publications

A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry

A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry

Differences in the half‐lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity

The liver and intracellular digestion: How liver cells eat!

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