Quantitative Struktur-Funktions-Korrelation an BiomembranenQuantitative Correlation of Structure and Function on Biomembranes

Microscopical studies on cells or tissues vitally stained with Neutral red (NR) were hitherto almost invariably confined to a few objects that could be investigated without tissue sectioning, for instance tissue culture. For NR, a cationic dye (molecular weight 289, PK 6,75) is easily soluble in water and organic solvents and diffuses during histological preparation for paraffin sectioning and even from cryostat sections. Thus comprehensive studies of vital staining of laboratory animals such as mouse and rat don't exist yet.This explains why it still remains doubtful, wether NR stains ‘preexisting’ or ‘newly formed’ granules, ‘paraplasmic’ cytoplasmic inclusion bodies or - complete or only partially - lysosomes and why it does so. We tried to solve these problems. Moreover we found that after intravital injection of the dye NR ‘stained’ the cells of the APUD-series (PEARSE) rather selectively as do catecholamines or catecholamine precursors. So it was to follow up wether NR could serve as a model for distribution studies of biogenic amines, too.The histological method that allows the localization of intravitally injected NR in tissue sections is freeze-drying. We applied freeze-drying to cryostat sections. The main advantages of this modiftcation are both the short time needed for the drying procedure and the large number of different tissues that can be cut and frozen-dried in the same time. In this way nearly all organs and tissues of the rat could be investigated.1 min after the intravenous injection of the dye NR has disappeared from the blood - at least in concentrations that are demonstrable in tissue sections. By using fluorescence microscopy the dye instead can be localized in all tissues being investigated mainly intracellularly. Apart from the diffuse staining of the cytoplasm in some tissues stained cell nuclei are observed. Intensely coloured nuclei together with a diffuse to scattered staining of the cytoplasm are signs of cell death.In some endocrine cells - mostly belonging to the APUD-series - a strong, often granular, reddening of the cytoplasm is seen; the nuclei are not stained. While the dosis and the form of application necessary to stain these endocrine cells, the intracellular localization and even the reactive groups (presumably carboxylic groups of the granule matrix) to which NR and catecholamines and their precursors are bound seem to be rather identic, some essential differences do exist: NR is easily and rapidly taken up and stored for a relatively short period, whereas biogenic amines accumulate in APUD - cells by active transport in a time - consuming process; their precursors are only stored following decarboxylation. - Secretory granules of some exocrine gland cells, too, may be vitally stained by NR.NR-staining of lysosomes is a well-known fact. We can add some details: a) There are-very characteristic for each of the tissues investigateddifferences in the time needed to stain lysosomes as well as in the duration of lysosomal staining: For instance lysosomes in the kidney proximal convolution are stained very rapidly directly after the injection of the dye and are destained in about 24 h, lysosomes in the thyroid epithelium are rapidly stained and destained, lysosomes in other organs need 45 to 60 min to get stained. c) There is no correlation between the vital staining of lysosomes and the characteristics lysosomes exhibit when stained by histologicalhistochemical methods in tissue sections. This may be due to the extraction from the tissue section of that particular component that, intravitally, binds the dye to lysosomes. - Differences in the composition of the lysosomal matrix in various organs are discussed as one major point of the heterogeneity of lysosomal vital staining with cationic dyes. d) NR vital staining of lysosomes of younger animals is less, that of older animals much more pronounced; lipopigment may, but must not neccessarily do so, exhibit a strong binding capacity for the cationic dye. e) In dehydration experiments the binding capacity oflysosomes is stronger, after premedication with reserpine less pronounced than normally. Desmethylimipramin has no effect at all.The so-called NR-crinom appears only in a few organs, resulting from autophagic as well as from heterophagic cell activity.Following albumin injection enlarged lysesomes are stained vitally in the renal proximal convolution. ‘Vacuoles’ induced by premedication with Macrodex® and glycerol remain unstained.NR is concentrated in the urine of the distal nephron and in the gastric lumen. Reabsorbtion occurs in the distal intestine.Essentially two factors are believed to be responsible for the pattern of NR vital staining: a) Its solubility that explains the distribution of the dye all over the organism, its diffusion through cell membranes and its elimination from the organism by ‘Non-ionic diffusion’. b) Its qualities as a light cationic dye that cause its - electrostatical - binding (‘storage’) to anionic sites of the tissue, for instance to carboxylic groups of the secretory granules of the APUDcells and carboxylic and/or phosphate groups of the lysosomal matrix.     

Uptake of lithium carmine by sinusoidal endothelial and Kupffer cells of the rat liver: new insights into the classical vital staining and the reticulo-endothelial system

Sinusoidal cells in the rat liver were studied in vivo and in vitro using the original vital staining with lithium carmine, which has contributed much to the development of the concept of the reticulo-endothelial system. Immunohistochemical and electron-microscopic studies revealed that the dye-incorporating cells were sinusoidal endothelial cells, Kupffer cells, and monocytes. The endothelial cells took up much more dye than did the Kupffer cells and bulged largely into the sinusoidal lumen. Electron microscopy revealed that small particles of lithium carmine were associated with coated vesicles of endothelial cells and ruffled membranes of Kupffer cells. In the endothelial cells, these particles were present in various concentrations within vacuolated structures and condensed in the lysosomes forming large aggregates of lithium carmine lumps. These lumps showed crystalline structures, within which the size of the individual particle was up to 30 nm in width and 50 nm in length. A few endothelial cells containing abundant dye underwent degeneration, and some were taken up by Kupffer cells. Liver endothelial cells isolated from lithium carmine-administered rats endocytosed fluorescence-labeled collagen. Isolated endothelial cells from normal rat liver, when cultured with lithium carmine, did not take up any dye, and their endocytosis of formaldehyde-treated albumin was inhibited dose-dependently. We conclude that in the liver, endothelial cells, but not Kupffer cells, predominantly take up lithium carmine. Furthermore, we propose the existence of a generalized cell system based on its vital staining capacity.     

Effects of Ph on Post-Mortem Retention of Trypan Blue Vital Staining in Tissues of Mice

Vital staining of aortas from mice injected subcutaneously (daily for 5 days) with trypan blue was studied. In routine paraffin sections elastic membranes were observed to be well stained and other medial elements unstained following fixation in 10% formaldehyde (25% formalin) at pH 7-9. An identical pattern of vital staining was observed in specimens that had been immersed for 48 hr in saline solutions at pH 7-11. Elastic membranes were not stained, but intermembranous connective tissue was stained after the following: (1) fixation in 10% formaldehyde at pH 1-4 and in Lavdowsky's solution (ethanol, formaldehyde, water and glacial acetic acid), pH 2.3-2.8; and (2) immersion in saline for 48 hr at pH 14. Aortic elastic membranes were vitally stained after fixation by intracardiac perfusion with 10% formaldehyde (pH 7-8) but not after perhion with Lavdowsky's fixative (pH 2.3-2.8). Vital staining was limited to medial elastic membranes in sections of fresh aorta made in a cryostat or by a regular freezing microtome. The vital staining (coarse cytoplasmic granules of dye) within macrophages (Kupffer cells and others) and in cytoplasm of renal tubular epithelium was well demonstrated following use of all methods discussed above     

Characterization of Merkel cells and mechanosensory axons of the rat by styryl pyridinium dyes

Summary The epidermal Merkel cells and their sensory innervation serve tactile sensation in vertebrates. In this study the fluorescent cationic mitochondrial dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), which has recently been used as a vital stain for motor and autonomic nerve terminals, was tested for its ability to stain Merkel cells and sensory fibers in the snout of the rat. Brightly-fluorescent structures resembling Merkel cells as well as nerve fibers and their terminations were evident in whole mounts of the vibrissal follicle. Unilateral denervation of the vibrissal follicles soon after birth resulted in a staining pattern remarkably similar to that obtained after labelling of the Merkel cells selectively with the fluorescent marker quinacrine, but all fiber staining was abolished. Likewise, in the separated epidermis of other skin regions, including the hairy and glabrous skin of the nose, the staining pattern revealed by 4-Di-2-ASP was indistinguishable from that obtained by quinacrine fluorescence. These results indicate that certain styryl pyridinium dyes may be used as vital stains for epidermal Merkel cells as well as cutaneous mechanosensory axons.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

Evaluation of methods for determining 6-hydroxydopamine cytotoxicity

Summary The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 μg/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations. This work was supported by an NIH National Research Service Award GM07204 to E. T. C., a gift from the Lola-Wright Foundation, NINCDS Grants NS14034 and NS15234, Robert Welch Grant H698, and an RCDA (NS00213) to J. R. P.     

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.     

Aqueous solutions of some basic dyes--their use in the cytochemical detection of DNA

The paper contains results of staining DNA-aldehyde molecules with aqueous solutions of brilliant cresyl blue, thionin or neutral red, following Feulgen procedure and also reports on the use of aqueous solutions of these dyes, with primary amino group(s) in their molecules, for staining animal tissue nuclei after extraction of RNA with cold phosphoric acid. The pH of the dye solutions most suitable for optimum staining is 6.0. The time necessary for optimum staining of DNA-aldehydes and DNA-phosphate groups are 10 and 2 min respectively for tissues fixed in formalin, paraformaldehyde or Craf. Tissue fixed in Buin-fluid stain slower. The absorption curves of nuclei stained for DNA-aldehyde molecules and DNA-phosphate groups, stained with each of the three dyes are different from each other. The in vitro absorption curves of aqueous solutions of the three dyes have also been presented. Some implications of the results obtained are discussed.     

Renal amino acid transport in immature and adult rats during chromate and cisplatinum-induced nephrotoxicity

Summary. The effects of sodium dichromate (chromate; 1 mg/100 g b. wt. s.c.) and cisdiamminedichloroplatinum(II) (CP; 0.6 mg/100 g b. wt. i.p.) on renal amino acid excretion and plasma amino acid composition were investigated in 10- and 55-day-old anaesthetised rats. On the basis of diuresis experiments on conscious rats the mentioned doses and times (1^st day after chromate in both age groups and in 10-day-old rats after CP and 3^rd day after CP in adult rats) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. Interestingly, in conscious 10-day-old rats chromate nephrotoxicity is not detectable after 1 mg/100 g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of chromate and CP in conscious rats. Urine volumes are lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. But GFR is significantly lower in 10-day-old rats, both in controls and after CP, whereas after chromate GFR is significantly reduced only in adult rats and age differences disappeared. In principle the renal fractional excretion (FE) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. Nevertheless, the amino acid plasma concentrations were relatively high in immature rats. However, both chromate and CP did not distinctly influence amino acid plasma concentrations. But in both age groups the administration of chromate and CP significantly decreased amino acid reabsorption capacity (increase in FE) as a sign of nephrotoxicity, most pronounced in adult rats after CP. The investigation of renal amino acid handling confirms (1) that both CP and chromate are nephrotoxins, (2) that CP was more nephrotoxic in 55-day-old animals compared to immature rats as could be demonstrated before using other parameters for nephrotoxicity testing and showed (3) that determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, especially in immature rats. Received March 3, 2000 Accepted October 11, 2000     

Cytophotometric determination of the binding of basic dyes by DNA following acetylation

Summary DNA was removed from various tissues by histochemical acetylation of amino groups in proteins using pure acetic anhydride, as demonstrated by cytophotometric (UV, Feulgen, gallocyanin chromalum) and biochemical techniques. Since new phosphate groups were simultaneously exposed, the intensity of methylene blue staining was increased in spite of the nucleic acid release. Under conditions where no extraction occurs the staining intensity increases for more than 30 per cent. On the other hand, the staining intensity of gallocyanin chromalum kept constant. As it had been demonstrated previously, that gallocyanin chromalum binds to about 86 per cent of the DNA phosphate groups, it was concluded that this dye binds to a higher percentage of phosphate groups than do the usual basic dyes. Since it is not possible under the conditions used to make all nucleic acid phosphate groups available for basic dye binding by blocking the amino groups of proteins it can be assumed that not only electrostatic, but also spatial and steric relationships influence the binding capacity of basic dyes to the phosphate groups of nucleoproteins.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.     

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

Ammoniacal silver method in alkaline pH range as a technique for detection of morpho-functional features of nerve tissue elements]

An alkaline range pH 9.1-12.3 instead of values pH 7.0-7.1 was used to localize lysine-rich and arginine-rich nucleoproteins by ammonia silver (AS) staining method (Black and Ansley, 1966) in brain structures of rabbits and rats. This modification of AS method made it steady to reveal lysine-rich and arginine-rich nucleoproteins and was able to demonstrate the high variability of protein localization in relation to the balance of essential amino acids in the brain structures with different functions. The best results of morphological staining of nerve, glial cells and their fibres were found at pH 10.6--the point of maximal dissociation of lysine--NH2 groups. At the same time the new modification of AS method allows the use of all alkaline range pH 9.1-12.3 for revealing the cells of nervous tissue and to vary the treatment of sections with silver ions.     

Vitalfluorochromierung von Chromosomen und Kernstrukturen in LM- und HeLa-Zellen mit neuen Acridinfarbstoffen

Zusammenfassung Es wird über die Fluorochromierung von Chromosomen und von Kernstrukturen (Nucleolus-assoziiertes Chromatin) in lebenden HeLa- und LM-Zellen (Mäusefibroblasten) mit neuen Acridinfarbstoffen berichtet. Die Farbstoffe haben in 9-Stellung des Acridingerüsts eine Aminoethylgruppe mit verschiedenen Endgruppen (Formelschema). Fehlt der 9-Substituent, so werden bei Einwirkung des Farbstoffs nur Lysosomen gebildet. Besonders bewährt hat sich bei der Vitalfluorochromierung von Chromosomen und Kernchromatin der Farbstoff 3-Amino-6-methoxy-9-(2-hydroxyetylamino)acridin 1. Konzentrationen von 10^–3 M können bei der Vitalfärbung angewendet werden. Durch Messung des Sauerstoffumsatzes konnte gezeigt werden, daß die Atmungsaktivität der Zellen selbst bei diesen hohen Konzentrationen durch den Farbstoff nicht beeinflußt wird. Daraus schließen wir, daß wir tatsächlich Vitalfärbungen und nicht Postvitalfärbungen beobachtet haben. Ähnliche Eigenschaften hat der bekannte Vitalfarbstoff Acridinorange.

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Fluorescent staining of chromosomes and nuclear structures in living LM- and HeLa-cells with new acridine dyes

Summary Fluorescent staining of chromosomes and nuclear structures (nucleolus associated chromatin) in living HeLa- and LM-cells (mouse fibroblasts) with new acridine dyes is reported. The dyes have aminoethylgroups in 9-position with different endgroups at this residue (scheme of structures). Dyes without these 9-substituents only induce the formation of lysosomes. An exceptional position on vital staining of chromosomes and nuclear chromatin has the dye 3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine 1. Concentrations of 10^–3 M can be used in vital staining experiments. Measuring the consumption of oxygen we could demonstrate that the dye has no effect on the activity of respiration even at these high dye concentrations. Therefore we conclude that we have really observed vital staining and not postvital staining of chromosomes and nuclear chromatin. Similar properties has the well known vital dye acridine orange.

     

An assessment of methods used for measuring the recovery of mammalian cells from freezing and thawing

Traditional methods of determining cell viability (vital dye staining and plating efficiency) as a measure of recovery from freeze-thaw cycles were found particularly unsuitable for human diploid cell lines. A modified plating efficiency method (RI) and a method using the release of radiochromate from injured or dead cells were compared and assessed for their suitability as measurements of cell recovery from freezing and thawing.     

Vital Staining with Two Dis-Azo Textile Dyes

Subcutaneous injections of 0.25% saline solutions of two dis-azo textile dyes, calcodur pink 2BL, C. I. 353, also known as benzo fast pink 2BL and amidine fast rose 2BL, and a blue dye, dianil blue G, C. I. 508, were made on alternate days on albino rats for one week. The blue dye is closely similar to Niagara blue 4B, C. I. 520, and dianil blue R, C. I. 465. Staining reactions were much like those of other vital blue disazo dyes. Although the pink dye exhibited a similar staining pattern, there were notable differences. The tissues of most glands were stained pink or red. Nuclei of the tubular epithelial cells of the kidney contained red granules as did the cytoplasm of the Kupfer cells. Most unusual was the bright red staining of the elastica interna of medium and large sized arteries.     

An in vitro preparation to access cellular and neuronal components in the mouse inner ear

An in vitro mouse temporal bone preparation has been developed in order to allow the investigation of structures and functions in a living hearing organ. Fluorescent vital probes (a potentiometric styryl dye, RH 795, and a vital dye staining cellular cytoplasm, calcein) were perfused through the scala tympani to stain cellular components of the cochlea. Observations of the cochlear apical turn were performed using a confocal microscope. In spite of the anatomical constraints due to the small size of the mouse cochlea, detailed images were obtained. The sensory cells as well as their innervating nerve fibres were clearly seen. Nerve fibres crossing the tunnel of Corti and projecting to the outer hair cells could also be visualised.     

Neutral red uptake and clonogenic survival assays of the hyperthermic sensitization of tumor cells to tumor necrosis factor

Vital dye uptake and postfixation dye assays have recently been used to examine the interaction between short-term (24-48 h) exposures to the monokine, tumor necrosis factor (TNF), and hyperthermic treatments with the finding that synergistic increases in cytotoxicity occurred. However, survival measured by these short-term dye assays is not necessarily closely related to eventual loss of clonogenic capacity. Treatment-induced growth delays, delayed cytotoxic effects, or perturbations of vital dye sequestration mechanisms could result in a different measurement of surviving fraction than given by a clonogenic assay. In this study we directly compared the neutral red vital dye uptake and clonogenic survival assays and confirmed in both assays that TNF-sensitive (L-929) and TNF-resistant (EMT-6) phenotypes show greatly reduced survival when treated with combined recombinant human TNF (1.0-0.0005 micrograms/ml) and hyperthermia (1-2 h at 43 degrees C). Moreover, we confirmed that sensitization of the TNF-resistant EMT-6 cells was largely dependent on monokine treatment before hyperthermia and was reduced by the reverse sequence. The greatest sensitization of TNF-responsive L-929 cells also occurred when TNF treatment preceded heating. These results for clonogenic survival are consistent with the hypothesis that hyperthermia used in combination with TNF in vivo is more cytotoxic than TNF or hyperthermia separately.     

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells,cell suspensions,and primary monolayer cultures

Summary Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2±1.5×10⁻⁶ IU assayed at 37°C for 25 preparations of isolated hepatocytes. The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells. This study was supported in part by Grants HL-11945-11 and 1-RO1-AM 26520-01A1 from the National Institutes of Health, Bethesda, MD.     

Flow-cytometric determination of glutathione alterations in vital cells by o-phthaldialdehyde (OPT) staining

A flow-cytometric method for the detection of changes of cellular glutathione content in vital cells is described. The reaction is based on formation of a fluorescent product between o-phthaldialdehyde (OPT) and reduced glutathione (GSH). OPT is a more GSH-specific dye than other thiol-specific dyes (e.g., bromobimanes), because it forms a cyclic compound with GSH. Changes of GSH induced by oxidation or thiol-blocking agents are visualized in vital cells after a 5-min staining at room temperature.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.