Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles.

We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.     

Crude liver membrane fractions as substrate preserve liver-specific functions in long-term,serum-free rat hepatocyte cultures

Summary Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum—crude membrane fractions prepared from the liver of the same rat strain—was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-de-ethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Prevention of nitrofurantoin-induced cytotoxicity in isolated hepatocytes by fructose

Nitrofurantoin is a widely utilized urinary antimicrobial drug which has been associated with pulmonary fibrosis, neuropathy, and hepatitis as well as hemolytic anemia in glucose-6-phosphate dehydrogenase-deficient individuals. Incubation of freshly isolated rat hepatocytes with nitrofurantoin caused oxygen activation as a result of futile redox cycling. Glutathione disulfide (GSSG) was formed and rapidly exported from the cell resulting in complete glutathione (GSH) depletion followed by cell death. However, fructose prevented the export of GSSG from the cell and GSH levels recovered rapidly without cytotoxicity occurring. Fructose did not affect nitrofurantoin metabolism but rapidly depleted cellular ATP levels by approximately 80% which remained depressed during the incubation period. Fructose, however, did not protect hepatocytes from nitrofurantoin-induced cytotoxicity if GSH was depleted beforehand. Protection by fructose only occurred at concentrations which caused ATP depletion. These results suggest that fructose prevents nitrofurantoin-induced toxicity by depleting ATP and thereby preventing the ATP-dependent GSSG efflux. GSSG is retained enabling NADPH and glutathione-reductase to reduce the GSSG back to GSH, thereby protecting the cell from nitrofurantoin-induced oxidative stress.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Characteristics of taurine transport in rat hepatocytes in primary culture

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.     

Hormonal regulation of glutathione efflux

The efflux of GSH has been shown previously to be a saturable process in both isolated rat hepatocytes and perfused liver, suggesting a carrier-mediated transport mechanism. The possibility in hormonal regulation of this process has been raised by recent reports. Our present work examined the role of hormones known to affect intracellular signal transduction mechanisms on GSH efflux in cultured rat hepatocytes and perfused rat livers. We found that cAMP-dependent factors, such as cholera toxin (CT), dibutyryl cAMP, forskolin, and glucagon all stimulated GSH efflux in cultured rat hepatocytes. The efflux kinetics were compared in cultured cells incubated with or without CT; the stimulation of GSH efflux was related to a near doubling of the Vmax while exhibiting no significant alteration of the Km. The increase in intracellular cAMP level associated with the threshold for this stimulatory effect was 25% above control. The stimulatory effect of CT could not be blocked by cyclohexamide pretreatment or reversed by colchicine treatment. The stimulatory effect of glucagon was abolished in the presence of ouabain but not in the presence of barium. On the other hand, hormones which act through Ca2+ and protein kinase C, such as phenylephrine and vasopressin, had no effect on GSH efflux in the cultured cells. In the perfused liver model, glucagon (10 nM) and dibutyryl cAMP (8 microM) stimulated sinusoidal GSH efflux to 130 and 144% of control values, respectively, and increased bile flow while not affecting biliary GSH efflux. Finally, the physiological significance of glucagon-mediated stimulation of sinusoidal GSH efflux was assessed by both plasma GSH and glucose levels in response to in vivo glucagon infusion. The threshold dose of glucagon for significant increase in plasma GSH (5.21 pmol/min) was lower than for glucose (15.61 pmol/min). At the highest glucagon infusion rate (261 pmol/min), plasma GSH level doubled while glucose level increased 80%. In conclusion, increased cAMP stimulates GSH efflux in cultured rat hepatocytes and perfused livers. The stimulatory effect of cAMP is exerted at the sinusoidal pole and appears to be mediated by hyperpolarization of hepatocytes by stimulation of Na(+)-K(+)-ATPase. In vivo studies confirmed the importance of cAMP-mediated stimulation of sinusoidal GSH efflux as it resulted in significant elevation of the plasma GSH level.     

Culture duration alters the glutathione content and sensitivity to ethacrynic acid of rat hepatocyte monolayer cultures

Many of the differentiated functions of hepatocytes are lost in culture, yet addition of certain medium supplements can aid in the retention of differentiated character. Therefore, the effect of time in monolayer culture on rat hepatocyte glutathione (GSH) synthesis and sensitivity to the GSH detoxicated xenobiotic ethacrynic acid was examined in cultures with and without medium supplementation by transferrin and sodium selenite. GSH content was found to be about 12 nmol/µg DNA at 4 hr in culture and to approximately triple by 24 hr. Intracellular GSH levels continued to increase in transferrin/sodium selenite-supplemented cultures, from 32 to 41.6 nmol/µg DNA, while GSH levels in unsupplemented cultures declined to 18 nmol/µg DNA. However, the rate of GSH synthesis after diethylmaleate depletion was found to decrease from 4.2 to 2.8 nmol/hr/µg DNA at 4 and 24 hr after inoculation, respectively. GSH repletion rate increased to 3.9 nmol/hr/µg DNA at 48 hr. The GSH accumulation rate after depletion in supplemented cultures did not vary significantly over the initial 48 hr. Incubation for 3 hr with 100 µM ethacrynic acid (EA) did not elicit an increase in LDH leakage in hepatocyte monolayers after 4 or 48 hr in culture or in cultures with supplemented medium at any time point tested. Cultures 24 hr in medium without transferrin/sodium selenite supplementation exhibited significant LDH leakage after 3 hr of EA treatment. Over the 3 hr EA treatment, intracellular GSH content was decreased in all cultures. Only in the 24 hr unsupplemented cultures did GSH depletion exceed the 90% level previously associated with depletion of the mitochondrial pool of GSH and EA toxicity in hepatocytes. The experiments show that during the redifferentiation of hepatocytes in culture, a transient period occurs when apparent GSH synthesis is depressed and enhanced sensitivity to GSH-detoxicated compounds is observed. This period of increased sensitivity is prevented or at least delayed by inclusion of supplemental transferrin and sodium selenite, suggesting that redifferentiation can be regulated by extracellular influences.Abbreviations CYSSG cysteine-glutathione mixed disulfide - DEM diethyl maleate - EA ethacrynic acid - GSH reduced glutathione - GSSG oxidized glutathione - HBS HEPES buffered saline - HWME hepatocyte Williams' Medium E (WME with insulin, corticosterone and 0.5 mM methionine) - LDH lactate dehydrogenase - TS-HWME transferrin/sodium selenite-supplemented HWME - WME Williams' Medium E     

Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes

Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.     

Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis

We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (M_r cut-off 3000 Da). The analysis was performed at a constant temperature (28°C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 μM for GSH and 1.2 μM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1±2.6 and 0.45±0.15 (nmol/mg protein±S.D.), respectively. The ratio of GSH to GSSG was 17.8±1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification.     

Cystine uptake by cultured cells originating from dog proximal tubule segments

Summary Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1∶36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37°C with 0.025 mM [³⁵S]l-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of a) the intricacies of cystine metabolism and b) regulation of 1) the cystine-dibasic amino acid co-transporter system and 2) the development of the cystine-glutamate anti-porter system. Supported by National Institutes of Health, Bethesda, MD, grant no. DK40555 and The National Kidney Foundation of the Delaware Valley.     

Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes

The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (approximately 10%) was recovered by reductive treatment of cell protein with NaBH4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (greater than 200 microM) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione.     

Taurocholate uptake by adult rat hepatocytes in primary culture

Adult rat hepatocytes were cultured on Petri dishes for 25--30 h prior to measuring their ability to transport taurocholate. A rapid uptake of the bile acid (25 muM) was observed: about 20% was accumulated in the cells within 15 min. The taurocholate transport was saturable with an apparent Km of 28 +/- 10 muM and a maximal velocity V of 0.07 +/- 0.02 nmol/(micrograms DNA x min). Uptake was shown to be energy dependent as it was inhibited about 65% by antimycin A (20 micrograms/ml). The monohydroxylated bile acid taurolithocholate and the dihydroxylated taurochenodeoxycholate inhibited taurocholate transport to about 30 and 40% resp. of the control. The transport process was strongly dependent on sodium ions. It is concluded that the characteristics of taurocholate uptake into adult rat hepatocytes are very similar either in freshly prepared cells or in hepatocytes which are cultured on Petri dishes for 25--30 h.     

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles,Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.     

Properties of peroxisomes and their induction by clofibrate in normal adult rat hepatocytes in primary culture

Summary Alterations in peroxisomes and catalase activity and their responsiveness to clofibrate in adult rat hepatocytes in primary culture were investigated. The numbers of peroxisomes with and without crystalloid nucleoids per unit cytoplasmic area were preserved in cultured hepatocytes for 2 d after seeding at a level comparable to that of freshly isolated hepatocytes. At Day 3 in culture, the number of anucleoid peroxisomes was reduced in untreated hepatocytes, accompanied by more significant reduction in the number of nucleoid-containing peroxisomes, which decreased until Day 5. Peroxisome diameters were reduced in untreated hepatocytes at Day 2 and this decrease in the diameter was continued until Day 7. Catalase activity in untreated hepatocytes decreased markedly with culture age. The number of anucleoid peroxisomes was significantly greater in hepatocytes treated with 2mM clofibrate in culture than in freshly isolated hepatocytes for 2 d or in untreated hepatocytes of the same culture age through 7 d. The number of nucleoid-containing peroxisomes in the treated cells began to decrease in 3 d, but was greater than that of untreated cells at Days 3 and 5. Peroxisomes with well-developed nucleoids were observed frequently in the treated cells even at Day 7. Peroxisome diameters were greater in the treated cells than in untreated cells at Days 3, 5, and 7. Catalase activity was always higher in the treated cells than in untreated cells. These results suggest that clofibrate is effective in inducing peroxisome proliferation as well as in maintaining the organelles in cultured hepatocytes. This work was supported in part by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture, Japan, 448143, 50168, 501069, and 577196, and by a Grant-in-Aid from Hokkaido Geriatrics Research Institute.     

Metabolic response to anisoosmolarity of rat skeletal muscle in vitro.

Isolated rat hepatocytes exhibit an insulin-like anabolic response to hypoosmotic incubation and a glucagon-like catabolic response to hyperosmotic incubation. Recently, a distinct glycogenic response to hypoosmotic treatment was likewise reported for cultured rat myotubes. The present study examines the effects of anisoosmolar exposure on glucose metabolism in freshly isolated rat soleus muscle strips. Under the same experimental conditions as used for cultured myotubes, hypoosmolarity reduced net glycogen synthesis to 52%, while hyperosmolarity stimulated glycogen storage to 231% of isoosmolar control (nmol glucose incorporated into glycogen g(-1) x h(-1): hypoosmolar, 34+/-3; isoosmolar, 65+/-8; hyperosmolar, 150+/-11; p<0.01 each vs. isoosmolar). The responses of native skeletal muscle to anisoosmolarity are therefore in opposition to what has been described for hepatocytes and cultured myotubes. Further experiments on rat skeletal muscle revealed that the observed lack of a glycogenic response to hypoosmolarity persisted independent of medium composition, specifically with regard to prevailing glucose and K+ concentrations. In conclusion, hypoosmotic exposure inhibits glycogen synthesis in isolated rat soleus muscle, which clearly argues against the hypothesis that osmotic changes and cell swelling may be physiologically relevant stimulators of muscle glycogen synthesis.     

Cellular effects of N(4-ethoxyphenyl)p-benzoquinone imine, a p-phenetidine metabolite formed during peroxidase reactions

The interaction of N-(4-ethoxyphenyl)p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated.

When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea).

Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI.

NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 μM extensive bleb formation was followed by loss of cell membrane integrity and cell death.

To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.