Chemically induced antioxidant-sensitive respiration. Relation to glutathione content and lipid peroxidation in the perfused rat liver

The interrelations between the hepatic chemically induced antioxidant-sensitive respiration and the contents of malondialdehyde (MDA) and of reduced glutathione (GSH), were studied in the isolated hemoglobin-free perfused rat liver. Antioxidant-sensitive respiration was induced by the infusion of agents such as ethanol, iron, xanthine or t-butyl hydroperoxide, or by phenylhydrazine pretreatment in vivo. The development of this respiratory component occurred concomitantly with high levels of MDA in the perfused livers, while those of GSH were diminished.     

Influence of lysine on urea cycle activity and orotate formation in the isolated perfused rat liver

There was a reversible inhibition of urea formation in the perfused rat liver caused by 2.25-27 mM lysine acting with a Ki of 10.8 mM in competition with ornithine. Urea formation in the presence of inhibitory concentrations of lysine ranged between 2.3 and 2.9 mumol X min-1 X (g, liver wet)-1 after addition of 1 mM of citrulline, argininosuccinate or arginine, whereas it amounted to 0.5 mumol X min-1 X (g, liver wet)-1 after addition of ornithine, showing that lysine inhibited the urea cycle between ornithine and citrulline. There was a rise of basal orotate formation of 0.03 +/- 0.02 mumol X h-1 X (g, liver wet)-1 towards a maximum of 0.6 +/- 0.04 mumol X h-1 X (g, liver wet)-1 after addition of 13.5 mM lysine, provided orotate utilization was blocked with allopurinol. Maximal rates of orotate formation were reached when ammonium concentrations exceeded 1 mM. We conclude that an inhibition of urea synthesis and a rise of orotate formation are caused by lysine in the isolated liver in vitro at rates observed in vivo. Hence, these metabolic alterations observed in the whole animal are most probably due to changes of liver metabolism.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.     

Antioxidant capacity of desferrioxamine in biological systems

The antioxidant capacity of desferrioxamine (DF) was investigated in three biological systems. The addition of DF to rat brain homogenates undergoing autoxidation elicited a concentration dependent inhibition of both oxygen uptake and chemiluminescence, with a median inhibitory concentration (IC50) of 0.52 microM. In this system, Fe3+-induced light emission was completely abolished at a DF/Fe3+ molar ratio of 0.6. In rat erythrocyte suspensions supplemented with t-butyl hydroperoxide, DF lengthened the induction period and decreased the rate of oxygen consumption, with an IC50 of 300 microM. Infusion of increasing concentrations of DF to the perfused rat liver elicited a progressive decrease in the rate of oxygen consumption, with no alterations in the mitochondrial respiration. This DF-sensitive respiration has a maximal value of 200 nmol/g of liver/min, with a half-maximal rate at 120 microM DF. These results indicate that DF behaves as an efficient antioxidant either under basal conditions or in chemically-induced oxidative stress, through Fe3+ chelating and/or free-radical scavenging effects.     

Energy-linked cardiac transport system for glutathione disulfide

The relationship between the rate of glutathione disulfide (GSSG) export and the energy state was studied in isolated perfused rat heart. The intracellular GSSG level was maintained at saturation for transport (7.5 nmol GSSG X min-1 X g heart-1) by continuous perfusion with 20 microM t-butyl hydroperoxide. GSSG release was substantially restricted upon the addition of inhibitors of mitochondrial respiration such as KCN, antimycin A or rotenone. In contrast, no effect was observed on GSSG release during potassium-induced cardiac arrest, although changes in oxygen consumption and coronary flow were similar to those observed with KCN. The dependence of the GSSG transport rate on the cytosolic free ATP/ADP ratio reveals that GSSG transport is half-maximal at (ATP/ADP)free approximately equal to 10. The capacity of GSSG transport was unchanged by infusion of epinephrine, norepinephrine or dibutyryl cyclic AMP.     

Gluconeogenesis predominates in periportal regions of the liver lobule

Rates of gluconeogenesis from lactate were calculated in periportal and pericentral regions of the liver lobule in perfused rat livers from increases in O2 uptake due to lactate. When lactate (0.1-2.0 mM) was infused into livers from fasted rats perfused in either anterograde or the retrograde direction, a good correlation (r = 0.97) between rates of glucose production and extra O2 uptake by the liver was observed as expected. Rates of oxygen uptake were determined subsequently in periportal and pericentral regions of the liver lobule by placing miniature oxygen electrodes on the liver surface and measuring the local change in oxygen concentration when the flow was stopped. Basal rates of oxygen uptake of 142 +/- 11 and 60 +/- 4 mumol X g-1 X h-1 were calculated for periportal and pericentral regions, respectively. Infusion of 2 mM lactate increased oxygen uptake by 71 mumol X g-1 X h-1 in periportal regions and by 29 mumol X g-1 X h-1 in pericentral areas of the liver lobule. Since the stoichiometry between glucose production and extra oxygen uptake is well-established, rates of glucose production in periportal and pericentral regions of the liver lobule were calculated from local changes in rates of oxygen uptake for the first time. Maximal rates of glucose production from lactate (2 mM) were 60 +/- 7 and 25 +/- 4 mumol X g-1 X h-1 in periportal and pericentral zones of the liver lobule, respectively. The lactate concentrations required for half-maximal glucose synthesis were similar (0.4-0.5 mM) in both regions of the liver lobule in the presence or absence of epinephrine (0.1 microM). In the presence of epinephrine, maximal rates of glucose production from lactate were 79 +/- 5 and 59 +/- 3 mumol X g-1 X h-1 in periportal and pericentral regions, respectively. Thus, gluconeogenesis from lactate predominates in periportal areas of the liver lobule during perfusion in the anterograde direction; however, the stimulation by added epinephrine was greatest in pericentral areas. Differences in local rates of glucose synthesis may be due to ATP availability, as a good correlation between basal rates of O2 uptake and rates of gluconeogenesis were observed in both regions of the liver lobule in the presence and absence of epinephrine. In marked contrast, when livers were perfused in the retrograde direction, glucose production was 28 +/- 5 mumol X g-1 X h-1 in periportal areas and 74 +/- 6 mumol X g-1 X h-1 in pericentral regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of non-transferrin-bound iron clearance by rat liver

Recent evidence suggests that the hepatic iron-loading characteristic of hemochromatosis may result in part from efficient hepatic clearance of non-transferrin-bound iron, which is increased in this disorder. However, this hypothesis assumes that hepatic clearance remains highly efficient despite excess iron stores. We therefore studied hepatic uptake of non-transferrin-bound iron in the single-pass perfused rat liver under varying conditions. Animals were iron loaded or depleted by dietary manipulation, but no changes in the efficiency of ferrous iron uptake or the kinetic parameters were seen (single-pass extraction, 59-74%; Km, 16-19 microM; Vmax, 30-32 nmol X min-1 X g liver-1). Added divalent zinc, cobalt, and manganese ions reversibly inhibited ferrous iron uptake and the inhibition by zinc was shown to be competitive. Uptake required calcium, was markedly temperature-sensitive (delta E = 14.3 Kcal/mol), and was relatively insensitive to inhibition of cellular energy metabolism. Particles consistent with ferritin cores were seen in lysosomes of hepatic parenchymal cells within 30 min of perfusion with ferrous iron. These results suggest that ferrous iron is cleared from plasma by a passive, saturable transport process that is not regulated by the iron content of the liver and that may be shared with other transition metal ions. Because clearance is highly efficient, increased levels of non-transferrin-bound iron in plasma may present the liver with an obligatory iron load resulting in progressive accumulation and toxicity.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

Cytotoxic ether phospholipids. Different affinities to lysophosphocholine acyltransferases in sensitive and resistant cells

Alkyllysophospholipids (ALP) which are 1-O-alkyl analogs of the cell membrane component 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) represent a family of new antitumor drugs. Susceptibility of cells to ALP is correlated to a selective inhibition of fatty acid incorporation into 1,2-diacyl-sn-glycero-3-phosphocholine in intact cells. This report examines oleoyl-CoA-1-acyl-GPC acyl-transferase activities in cell-free systems of ALP-sensitive methylcholanthrene-induced fibrosarcoma cells (MethA cells) and ALP-resistant bone marrow-derived murine macrophages (BMM phi). The specific activities for the oleoyl-CoA-1-acyl-GPC acyltransferases were 1.05 +/- 0.06 nmol X mg-1 X min-1 and 2.98 +/- 0.27 nmol X mg-1 X min-1, respectively. The kinetic parameters for 1-palmitoyl-GPC were Km = 16.6 microM, Vmax = 4.3 nmol X mg-1 X min-1 (BMM phi) and Km = 7.6 microM, Vmax = 2.0 nmol X mg-1 X min-1 (MethA cells). In the presence of 1-O-octadecyl-2-O-methyl racemic glycero-3-phosphocholine (ET-18-OCH3), one of the most potent cytotoxic ALP, the acyltransferase was dose dependently inhibited in MethA cells with a 50% inhibition concentration at 40 micrograms/ml. The BMM phi-acyltransferase was not affected up to 80 micrograms of ET-18-OCH3/ml. The kinetic parameters (Km' = 15.4 microM, Vmax' = 2.2 nmol X mg-1 X min-1) suggest that ET-18-OCH3 is a competitive inhibitor in MethA cells. Inhibitor constants for ET-18-OCH3, calculated from Dixon plots, were found to be 423 microM (BMM phi) and 13 microM (MethA cells) indicating a 33-fold larger affinity of ET-18-OCH3 to the MethA cells than to the BMM phi acyltransferase. From these data we assume that the inhibition of oleic acid incorporation into cellular phosphocholine during the antineoplastic action of ALP may be due to different affinities of the inhibitor to the 1-acyl-GPC acyltransferases in different cell types.     

Degradation of the 34 amino acid gastrin by rat tissue homogenates

Extracts of rat kidney contain an enzyme (gastrinase) that is highly specific for degradation of the 34 amino acid gastrin (G34). The Michaelis constant (Km) for kidney is 0.36 +/- 0.04 microM and the Vmax is 9.5 +/- 2.4 nmol X g-1 X min-1. Extracts of liver and brain also have gastrin degrading activity but the enzymes responsible appear to be different from the kidney gastrinase. Km for the liver enzyme is 0.08 +/- 0.02 microM but its Vmax (0.10 +/- 0.02 nmol X g-1 X min-1) is only 1% of the kidney gastrinase; Km for the brain enzyme is 0.10 +/- 0.03 microM but its Vmax (0.023 +/- 0.007 nmol X g-1 X min-1) is even lower than for the liver enzyme. The liver and brain enzymes appear to be less specific than the kidney enzyme with respect to competitive inhibition by insulin and glucagon. Cholecystokinin octapeptide is less inhibitory than the other peptides even though it shares a common C-terminal pentapeptide with G34. These findings are consistent with in vivo studies which have demonstrated that the dog kidney is an important site for extraction and degradation of endogenous dog gastrin but there is little or no hepatic removal of G34.     

Pathophysiological consequences of enhanced intracellular superoxide formation in isolated perfused rat liver.

The potential toxicity of enhanced intracellular reactive oxygen formation was investigated in isolated perfused livers of male Fischer rats. The presence of the redox-cycling agent diquat in the perfusate (200 microM) increased the basal efflux of glutathione disulfide (GSSG) into bile (2.65 +/- 0.26 nmol GSH-equivalents/min per g liver wt.) and perfusate (0.55 +/- 0.15 nmol/min per g) approximately 10-fold. Since no evidence was found for degradation of GSSG in the biliary tract of these animals, it could be estimated that diquat induced a constant O2- generation of approximately 1000 nmol/min per g liver wt for 1 h. Thus, reactive oxygen formation under these conditions was 1-2 orders of magnitude higher than under various pathophysiological conditions. Only minor liver injury (release of lactate dehydrogenase activity) was observed. To increase the susceptibility of the liver to the oxidant stress, animals were pretreated in vivo with 200 mg/kg body wt. phorone, which caused a 90% depletion of the hepatic glutathione content, 100 mg/kg ferrous sulfate, a combination of phorone and ferrous sulfate, or 40 mg/kg BCNU, which caused a 60% inhibition of hepatic GSSG reductase. Only the combined treatment of phorone + ferrous sulfate or BCNU caused a significant increase of the diquat-induced liver injury. Our results demonstrated an extremely high resistance of the liver against intracellular reactive oxygen formation (even with impaired detoxification systems) and can serve as reference for the evaluation of potential contributions of reactive oxygen to liver injury in various disease states.     

Adenine nucleotide synthesis from inosine during normoxia and after ischaemia in the isolated perfused rat heart

[14C]inosine in a range of concentrations of 20 microM to 1 mM was administered to the isolated perfused rat heart for 30 min. The incorporation of the nucleoside into myocardial adenine nucleotides increased for extracellular concentrations of the precursor up to 50 microM, reaching a plateau at 60 nmol . g-1 X 30 min-1 with concentrations ranging between 50 and 200 microM. The supply of 500 microM and 1 mM of inosine induced a further increase in cardiac adenine nucleotide synthesis to about 200 nmol . g-1 X 30 min-1. When supplied during low flow ischaemia (0.5 mL . min-1, 30 min.), 1 mM of inosine protected the heart against ATP degradation, while 100 microM of inosine was inefficacious. In the presence of 1 mM of inosine on reperfusion the adenine nucleotide content of the heart was similar to that observed in the absence of the nucleoside. The incorporation of [14C]inosine into adenine nucleotides was, in this last condition, below the value measured before ischaemia. Inosine administration was effective in protecting the heart against ischaemic breakdown of glycogen and favoured postischaemic restoration of glycogen stores.     

Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

Glutamine and glutamate transport by Anabaena variabilis

Anabaena variabilis, a dinitrogen-fixing cyanobacterium, has high- and low-affinity systems for the transport of glutamine and glutamate. The high-affinity systems have Km values of 13.8 and 100 microM and maximal rates of 13.2 and 14.4 nmol X min-1 X mg of chlorophyll a-1 for glutamine and glutamate, respectively. The low-affinity systems have Km values of 1.1 and 1.4 mM and maximal rates of 125 and 100 nmol X min-1 X mg of chlorophyll a-1 for glutamine and glutamate, respectively. Glutamine was unable to support growth of A. variabilis in the absence of any other nitrogen source, and glutamate alone at 500 microM was inhibitory to its growth. The analog L-methionine-DL-sulfoximine (MSX) was transported by a high-affinity system with a Km of 34 microM. Competition experiments and the transport characteristics of a specific class of MSX-resistant mutants imply that glutamine, glutamate, and MSX share a common component for transport. A second class of MSX-resistant mutants had a glutamine synthetase activity with altered affinity constants for glutamine and glutamate relative to the wild-type enzyme.     

Calcium ion fluxes induced by the action of alpha-adrenergic agonists in perfused rat liver.

Phenylephrine (2.0 microM) induces an alpha 1-receptor-mediated net efflux of Ca2+ from livers of fed rats perfused with medium containing physiological concentrations (1.3 mM) of Ca2+. The onset of efflux (7.1 +/- 0.5 s; n = 16) immediately precedes a stimulation of mitochondrial respiration and glycogenolysis. Maximal rates of efflux are observed between 35 s and 45 s after alpha-agonist administration; thereafter the rate decreases, to be no longer detectable after 3 min. Within seconds of terminating phenylephrine infusion, a net transient uptake of Ca2+ by the liver is observed. Similar effects were observed with vasopressin (1 m-unit/ml) and angiotensin (6 nM). Reducing the perfusate [Ca2+] from 1.3 mM to 10 microM had little effect on alpha-agonist-induced Ca2+ efflux, but abolished the subsequent Ca2+ re-uptake, and hence led to a net loss of 80-120 nmol of Ca2+/g of liver from the tissue. The administration at 5 min intervals of short pulses (90 s) of phenylephrine under these conditions resulted in diminishing amounts of Ca2+ efflux being detected, and these could be correlated with decreased rates of alpha-agonist-induced mitochondrial respiration and glucose output. An examination of the Ca2+ pool mobilized by alpha-adrenergic agonists revealed that a loss of Ca2+ from mitochondria and from a fraction enriched in microsomes accounts for all the Ca2+ efflux detected. It is proposed that the alpha-adrenergic agonists, vasopressin and angiotensin mobilize Ca2+ from the same readily depleted intracellular pool consisting predominantly of mitochondria and the endoplasmic reticulum, and that the hormone-induced enhanced rate of mitochondrial respiration and glycogenolysis is directly dependent on this mobilization.     

Changes in oxygen consumption induced by t-butyl hydroperoxide in perfused rat liver. Effect of free-radical scavengers.

The addition of t-butyl hydroperoxide to perfused rat liver elicited a biphasic effect on hepatic respiration. A rapid fall in liver oxygen consumption was initially observed, followed by a recovery phase leading to respiratory rates higher than the initial steady-state values of oxygen uptake. This overshoot in hepatic oxygen uptake was abolished by free-radical scavengers such as (+)-cyanidanol-3 or butylated hydroxyanisole at concentrations that did not alter mitochondrial respiration. (+)-Cyanidanol-3 was also able to facilitate the recovery of respiration, the diminution in the calculated rate of hydroperoxide utilization and the decrease in liver GSH content produced by two consecutive pulses of t-butyl hydroperoxide. It is suggested that the t-butyl hydroperoxide-induced overshoot in liver respiration is related to increased utilization of oxygen for lipid peroxidation as a consequence of free radicals produced in the scission of the hydroperoxide by cellular haemoproteins.     

Regulation of the glycine cleavage system in the isolated perfused rat liver

The catabolism of glycine in the isolated perfused rat liver was investigated by measuring the production of 14CO2 from [1-14C]- and [2-14C]glycine. Production of 14CO2 from [1-14C]glycine was maximal as the perfusate glycine concentration approached 10 mM and exhibited a maximal activity of 125 nmol of 14CO2 X g-1 X min-1 and an apparent Km of approximately 2 mM. Production of 14CO2 from [2-14C]glycine was much lower, approaching a maximal activity of approximately 40 nmol of 14CO2 X g-1 X min-1 at a perfusate glycine concentration of 10 mM, with an apparent Km of approximately 2.5 mM. Washout kinetic experiments with [1-14C]glycine exhibited a single half-time of 14CO2 disappearance, indicating one metabolic pool from which the observed 14CO2 production is derived. These results indicate that the glycine cleavage system is the predominant catabolic fate of glycine in the perfused rat liver and that production of 14CO2 from [1-14C]glycine is an effective monitor of metabolic flux through this system. Metabolic flux through the glycine cleavage system in the perfused rat liver was inhibited by processes which lead to reduction of the mitochondrial NAD(H) redox couple. Infusion of beta-hydroxybutyrate or octanoate inhibited 14CO2 production from [1-14C]glycine by 33 and 50%, respectively. Alternatively, infusion of acetoacetate stimulated glycine decarboxylation slightly and completely reversed the inhibition of 14CO2 production by octanoate. Metabolic conditions which are known to cause a large consumption of mitochondrial NADPH (e.g. ureogenesis from ammonia) stimulated glycine decarboxylation by the perfused rat liver. Infusion of pyruvate and ammonium chloride stimulated production of 14CO2 from [1-14C]glycine more than 2-fold. Lactate plus ammonium chloride was equally as effective in stimulating glycine decarboxylation by the perfused rat liver, while alanine plus ammonium chloride was ineffective in stimulating 14CO2 production.     

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Effects of gamma-irradiation on isolated rat liver mitochondria

Gamma-irradiation of isolated rat liver mitochondria with doses of up to 475 Gy leading to hydrated electrons (G = 1.9, corrected for reaction with solutes), 30 Gy leading to carbohydrate radicals, (G = 5.6), 100 Gy leading to superoxide radicals (G = 6.2), and 130 Gy leading to formate radicals (G = 6.2) showed, within the error of the measurements, no effects on the rate of oxygen uptake in the various respiratory states, the respiratory control ratio, or the adenosine diphosphate to atomic oxygen ratio. Typical values obtained were 0.020-0.100 nmol O2 s-1 mg protein-1 for State 1 respiration, 0.25-0.33 nmol O2 s-1 mg protein-1 for State 4 respiration and 0.65-1.10 nmol O2 s-1 mg protein-1 for State 3 respiration. Typical respiratory control ratios ranged from 2.0-3.5 for succinate and 4.0-6.5 for a 1:1 glutamate: malate substrate mixture. Adenosine diphosphate to atomic oxygen ratios with succinate as substrate varied from 1.6 to 1.9. Because these results are unexpected, in situ and in vitro irradiated mitochondria were examined in an electron microscope and compared to mitochondria in situ, non-irradiated mitochondria and mitochondria isolated after whole liver irradiation. Irradiation of isolated mitochondria with 375 Gy results in the partial destruction of the mitochondrial outer membrane with no significant changes in respiratory rates.     

Non-transferrin-bound iron uptake by rat liver. Role of membrane potential difference

Non-transferrin-bound iron is efficiently cleared from serum by the liver and may be primarily responsible for the hepatic damage seen in iron-overload states. We tested the hypothesis that transport of ionic iron is driven by the negative electrical potential difference across the liver cell membrane. Extraction of 55Fe-labeled ferrous iron (1 microM) from Krebs bicarbonate buffer by the perfused rat liver was continuously monitored as the transmembrane potential difference (measured using conventional microelectrodes) was altered over the physiologic range by isosmotic ion substitution. Resting membrane potential in Krebs bicarbonate buffer was -28 +/- 1 mV. Perfusion with 1 microM ferrous iron caused a reversible 3 +/- 1 mV depolarization, and higher concentrations of iron caused even greater depolarization. Conversely, depolarization of the liver cells consistently reduced iron extraction. Replacement of sodium with potassium (70 mM) or choline (131 mM) depolarized the hepatocytes to -15 and -20 mV and decreased iron extraction by 28 and 31%, respectively. Perfusion with bicarbonate-free solutions containing tricine buffer (10 mM) reduced the membrane potential to -23 mV and reduced iron extraction by 18%. In contrast, the high basal extraction of iron (91.1 +/- 1.4%) was not further increased by substitution of nitrate for chloride (-46 mV) or infusion of glucagon (-34 mV). All effects were reversible, suggesting that perfusion with 1 microM iron produced little toxicity. These findings are consistent with an electrogenic transport mechanism for uptake of non-transferrin-bound iron that is driven by the transmembrane potential difference.