Hypoxic damage generates reactive oxygen species in isolated perfused rat liver

The aim of the present study was to investigate the possible role of reactive oxygen species in the pathogenesis of hypoxic damage in isolated perfused rat liver. One hour of hypoxia caused severe cell damage (lactate dehydrogenase release of greater than 12,000 mU/min/g liver wt) and total irreversible cholestasis which was accompanied by a loss of cellular ATP and a marked decrease in lactate efflux. Tissue glutathione disulfide (GSSG) content and GSSG efflux as a measure of hepatic reactive oxygen formation was less than 1% of total glutathione before and during hypoxia. Upon reoxygenation, however, hepatic GSSG content increased sharply to about twice the control values and GSSG efflux increased several-fold to around 3-4 nmol GSH-equivalents/min/g. The release of lactate dehydrogenase decreased upon reoxygenation and tissue ATP content recovered partially. When livers were reoxygenated at an earlier time interval than 1 hr of hypoxia, i.e., before the onset of damage, no enhanced GSSG formation was observed. The results demonstrate that hypoxic damage is a prerequisite to reactive oxygen formation during the subsequent reoxygenation period. Thus, reactive oxygen species appear unlikely to play a crucial role in the pathogenesis of hypoxic liver damage in the hemoglobin-free, isolated perfused liver model.     

Effect of BCNU pretreatment on diquat-induced oxidant stress and hepatotoxicity

Diquat administration produces hepatic necrosis in male Fischer-344 rats, and minimally in male Sprague-Dawley rats, with massive oxidant stress observable in both strains as evidenced by increased biliary efflux of glutathione disulfide (GSSG). Pretreatment of both strains of rats with 80 mg/kg of 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU) inhibited hepatic glutathione reductase by 75 percent and increased dramatically the biliary efflux of GSSG produced by administration of diquat. BCNU pretreatment markedly potentiated diquat hepatotoxicity in the Fischer rats and modestly in Sprague-Dawley rats. BCNU-pretreated Fischer rats did not show an enhanced depletion of nonprotein sulfhydryls in response to diquat, in spite of the dramatic potentiation of the hepatic necrosis produced, nor were protein thiols depleted. The effects of BCNU on diquat hepatotoxicity in the Fischer rat are consistent with a critical role for reactive oxygen species in the pathogenesis of the observed hepatic necrosis and for the protective role of the glutathione peroxidase/reductase system. The data suggest that shifts in thiol-disulfide equilibria are not responsible for the cell death produced by oxidant stress in vivo, but are consistent with a role for lipid peroxidation in the pathogenesis of the lesion.     

Vascular Oxidant Stress and Hepatic Ischemia/Reperfusion Injury

The objective of this study was to test the hypothesis that the extracellular oxidation of glutathione (GSH) may represent an important mechanism to limit hepatic ischemia/reperfusion injury in male Fischer rats in vivo. Basal plasma levels of glutatione disulfide (GSSG: 1.5 ± 0.2μM GSH-equivalents), glutathione (GSH: 6.2 ± 0.4 μM) and alanine aminotransferase activities (ALT 12 ± 2U/I) were significantly increased during the l h reperfusion period following l h of partial hepatic no-flow ischemia (GSSG: 19.7 ± 2.2μM; GSH 36.9 ± 7.4μM; ALT: 2260 ± 355 U/l). Pretreatment with 1,3-bis-(2-chloroethyl)-I-nitrosourea (40mg BCNU/kg), which inhibited glutathione reductase activity in the liver by 60%. did not affect any of these parameters. Biliary GSSG and GSH efflux rates were reduced and the GSSG-to-GSH ratio was not altered in controls and BCNU-treated rats at any time during ischemia and reperfusion. A 90% depletion of the hepatic glutathione content by phorone treatment (300 mg/kg) reduced the increase of plasma GSSG levels by 54%, totally suppressed the rise of plasma GSH concentrations and increased plasma ALT to 4290 ± 755 U/I during reperfusion. The data suggest that hepatic glutathione serves to limit ischemialreperfusion injury as a source of extracellular glutathione, not as a cofactor for the intracellular enzymatic detoxification of reactive oxygen species.     

Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis

Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.     

Lethal injury by diquat redox cycling in an isolated hepatocyte model

Hepatocyte isolated by collagenase perfusion of livers of male Fischer-344 rats, and treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (50 microM for 30 min at 37 degrees C) to inhibit glutathione reductase, were significantly more vulnerable to cytotoxicity of the bipyridyl herbicide diquat than similarly treated cells of Sprague-Dawley rats. Without compromise of cell defenses by BCNU, diquat was not cytotoxic to hepatocytes from either strain. Microsomal enzyme induction with phenobarbital (80 mg/kg ip for 3 days before hepatocyte isolation) did not potentiate killing of Fischer hepatocytes by diquat. Specific activities of NADPH-cytochrome P-450 reductase in isolated Fischer and Sprague-Dawley rat liver microsomes utilizing 1 mM diquat as acceptor were 0.085 +/- 0.017 and 0.076 +/- 0.028 mumol/mg.min (mean +/- SEM, N = 5), respectively, indicating the capacity for very active redox cycling of diquat by this route in both strains. The serine protease inhibitor, phenylmethylsulfonyl fluoride (100 microM), had no effect on diquat cytotoxicity, but both leupeptin (100 micrograms/ml) and antipain (50 or 100 microM) were able to delay, through not completely prevent, diquat-induced cell death. The phospholipase inhibitors, chlorpromazine (50 or 100 microM) and dibucaine (50 or 100 microM), similarly delayed but did not prevent cell death. Diquat increased the rate of hepatocyte phospholipid hydrolysis, measured as release into the suspending medium of [14C]arachidonic acid previously incorporated into hepatocyte lipids, but although chlorpromazine decreased phospholipid hydrolysis to the control rate, only partial protection against diquat cytotoxicity was seen. These data suggest that activation of phospholipase A2 and proteases by elevation of cytosolic free Ca2+ cannot account entirely for the loss of cell viability observed in the presence of cytotoxic concentrations of diquat.     

Hyperoxia enhances lung and liver nuclear superoxide generation

Porcine lung and liver nuclei generated superoxide (O-2) at a rate which increased with increasing oxygen concentration. NADH-dependent O-2 generation increased from 0 to 2.21 +/- 0.11 nmol/min per mg protein for lung nuclei and from 0.16 +/- 0.09 to 1.34 +/- 0.14 nmol/min per mg protein for liver nuclei, when oxygen concentration increased from 0 to 100%. NADPH-dependent O-2 generation increased similarly in liver nuclei (from 0.20 +/- 0.09 to 1.20 +/- 0.12 nmol/min per mg protein), while lung nuclei produced only 0.45 +/- 0.09 nmol/min per mg protein at 100% oxygen. NADH and NADPH had an additive effect on O-2 generation by liver nuclei, yielding 2.58 +/- 0.21 nmol/min per mg protein at 100% oxygen. Very little or no superoxide dismutase activity was present in washed nuclear preparations. The oxygen-dependence of nuclear O-2 generation shows that nuclear-derived partially reduced species of oxygen may affect nuclear function during hyperoxia or other metabolic situations where overproduction of oxygen radicals is problematic.     

Chemically-induced glutathione depletion and lipid peroxidation

Malondialdehyde (MDA) formation in mouse liver homogenates was measured in the presence of various glutathione depletors (5 mmol/l). After a lag phase of 90 min, the MDA formation increased from 1.25 nmol/mg protein to 14.5 nmol/mg in the presence of diethyl maleate (DEM), to 10.5 with diethyl fumarate (DEF) and to 4 with cyclohexenon by 150 min. It remained at 1.25 nmol/mg with phorone and in the control. On the other hand, glutathione (GSH) dropped from 55 nmol/mg to 50 nmol/mg in the control to, < 1 with DEM, to 46 with DEF, to 3 with cyclohexenon and to 7 with phorone. The data show that the potency to deplete GSH is not related to MDA production in this system. DEM stimulated in vitro ethane evolution in a concentration-dependent manner and was strongly inhibited by SKF 525A. From type I binding spectra to microsomal pigments the following spectroscopic binding constants were determined: 2.5 mmol/l for phorone, 1.2 mmol/l for cyclohexenon, 0.5 mmol/l for DEM and 0.3 mmol/l for DEF. In isolated mouse liver microsomes NADPH-cytochrome P-450 reductase and NADH-cytochrome b₅ reductase activity were unaffected by the presence of DEM, whereas ethoxycoumarin dealkylation was inhibited. Following in vivo pretreatment, hepatic microsomal electron flow as determined in vitro was augmented in the presence of depleting as well as non-depleting agents, accompanied by a shift from O₂⁻ to H₂O₂ production. It is concluded that it is not the absence of GSH which causes lipid peroxidation after chemically-induced GSH depletion but rather the interaction of the chemicals with the microsomal monoxygenase system.     

Formation of activated oxygen in the hypoxic rat liver

The biliary GSSG efflux rate of normoxic perfused rat liver was 1.5 +/- 0.2 nmol/min/g liver wet weight. The GSSG efflux rate as indicator for the flux through the glutathione peroxidase reaction and, therefore, for an oxidative loading increased with the extent of hypoxia. 2.6 +/- 0.5 nmol/min/g were released from the severely hypoxic liver. The hydroxyl radical scavenger formate as well as the xanthine oxidase inhibitor allopurinol reduced the efflux rate of GSSG. GSH was released from the perfused liver at a rate of 15.5 nmol/min/g which was nearly unchanged in severe hypoxia. The high rate of glucose liberation from the hypoxic liver declined to almost that of the normoxic organ in the presence of formate. There is an 'oxidative stress' during hypoxic liver perfusion which probably originates from increased generation of activated oxygen species in the degradation of purine nucleotides.     

Effect of low flow ischemia-reperfusion injury on liver function

The release of liver enzymes is typically used to assess tissue damage following ischemia-reperfusion. The present study was designed to determine the impact of ischemia-reperfusion on liver function and compare these findings with enzyme release. Isolated, perfused rat livers were subjected to low flow ischemia followed by reperfusion. Alterations in liver function were determined by comparing rates of oxygen consumption, gluconeogenesis, ureagenesis, and ketogenesis before and after ischemia. Lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) activities in effluent perfusate were used as markers of parenchymal and endothelial cell injury, respectively. Trypan blue staining was used to localize necrosis. Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were measured in the perfusate as indicators of intracellular oxidative stress. LDH activity was increased 2-fold during reperfusion compared to livers kept normoxic for the same time period whereas PNP activity was elevated 5-fold under comparable conditions. Rates of oxygen consumption, gluconeogenesis, and ureagenesis were unchanged after ischemia, but ketogenesis was decreased 40% following 90 min ischemia. During reperfusion, the efflux rates of total glutathione and GSSG were unchanged from pre-ischemic values. Significant midzonal staining of hepatocyte nuclei was observed following ischemia-reperfusion, whereas normoxic livers had only scattered staining of individual cells. Reperfusion of ischemic liver caused release of hepatic enzymes and midzonal cell death, however, several major liver functions were unaffected under these experimental conditions. These data indicate that there were negligible changes in liver function in this model of ischemia and reperfusion despite substantial enzyme release from the liver and midzonal cell death.     

Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury

Hypoxia caused severe damage in isolated perfused livers from fasted male Fischer rats without evidence of the formation of reactive oxygen species during hypoxia. Reoxygenation caused a significant increase in intracellular oxygen species in the injured liver, as indicated by increases in sinusoidal GSSG efflux and tissue GSSG levels. Both parameters were elevated further by addition of KCN (100 microM) or antimycin A (8 microM). Sinusoidal GSSG efflux was suppressed in part by addition of allopurinol (500 microM) and enhanced by hypoxanthine (250 microM). Xanthine oxidase appears to be a partial source, and damaged mitochondria a continuous and quantitatively greater source, of reactive oxygen as a result of liver injury following hypoxia.     

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Diquat and paraquat are nonspecific defoliants that induce toxicity in many organs including the lung, liver, kidney, and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In this study, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese hamster ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild-type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective at generating ROS compared to paraquat (K(M)=1.0 and 44.2μM, respectively, for H(2)O(2) generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5μM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (V(max)≈6.0nmol H(2)O(2)/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than in CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than in CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.     

Some characteristics of hydrogen- and alkylhydroperoxides metabolizing systems in cardiac tissue

The effect of hydroperoxides on the cardiac tissue was studied by using hemoglobin-free perfused rat heart. Ethylhydroperoxide was degraded mainly through the glutathione peroxidase system of the heart at a maximal rate of about 1.2 mumol/min per g wet wt. When ethylhydroperoxide infused was not degraded completely, the hydroperoxide concentration in the effluent perfusate paralleled the formation of ferrylmyoglobin in the heart. The infusion of ethylhydroperoxide caused release of oxidized glutathione into the effluent perfusate as a result of the enhancement of the cytosolic glutathione peroxidase reaction. The leakage of oxidized glutathione reached the maximal rate of 3.5 nmol/min per g wet wt with the infusion of 175 microM ethylhydroperoxide. At hydroperoxide concentrations above 150 microM, oxidations of pyridine nucleotides and of cytochrome a + a3 occurred, probably through a stimulation of the mitochondrial glutathione peroxidase reaction, and resulted in sudden failure of the heart function. The infusion of t-butyl- and cumene-hydroperoxides, which are unable to react with myoglobin, also caused the oxidations of pyridine nucleotides and cytochrome a + a3, the inhibition of oxygen consumption and the failure of heart function. The results indicate that the cardiac toxicity of hydroperoxides is due mainly to their effect on mitochondrial metabolism.     

Cellular glutathione peroxidase protects mice against lethal oxidative stress induced by various doses of diquat

This study was to determine if cellular glutathione peroxidase (GPX1) protects against acute oxidative stress induced by diquat. Lethality and hepatic biochemical indicators in GPX1 knockout mice [GPX1(-/-)] were compared with those of wild-type mice (WT) after an intraperitoneal injection of diquat at 6, 12, 24, or 48 mg/kg of body weight. Although the WT survived all the doses, the GPX1(-/-) survived only 6 mg diquat/kg and were killed by 12, 24, and 48 mg diquat/kg at 52, 4.4 and 3.9 hr, respectively. Compared with those of surviving mice that were sacrificed on Day 7, the dead GPX1(-/-) had diquat dose-dependent increases (P < 0.05) in plasma alanine aminotransferase (ALT) activities. The GPX1(-/-) also had higher (P < 0.05) liver carbonyl contents than those of the WT, but the differences were irrespective of diquat doses. Whereas hepatic total GPX and phospholipid hydroperoxide glutathione peroxidase activities or hepatic GPX1 protein was not significantly affected by the diquat treatment, liver thioredoxin reductase and catalase activities were lower (P < 0.05) in the GPX1(-/-) injected with 12 mg diquat/kg than those of other groups. In conclusion, normal GPX1 expression is necessary to protect mice against the lethality, hepatic protein oxidation, and elevation of plasma ALT activity induced by 12-48 mg diquat/kg.     

Utilisation of triacylglycerol and non-esterified fatty acid by the working rat heart: myocardial lipid substrate preference

Utilisation and subsequent metabolic fate (oxidation; tissue lipid deposition) of non-esterified fatty acid (NEFA), very-low-density lipoprotein-triacylglycerol (VLDL-TAG), and chylomicron-triacylglycerol (CM-TAG) alone or in combination by isolated working rat heart were examined. Cardiac mechanical function was maintained regardless of lipid substrate used. NEFA and CM-TAG were assimilated to a greater extent than VLDL-TAG; CM-TAG utilisation (76+/-10 nmol fatty acid/min per g wet wt.; n=8), but not VLDL-TAG utilisation (16+/-2 nmol fatty acid/min per g wet wt.; n=8), was suppressed in the presence of NEFA, but TAG (CM or VLDL) did not alter NEFA utilisation (57+/-9 nmol fatty acid/min per g wet wt.; n=8). Most (about 75%) of the lipid utilised was oxidised. In the presence of NEFA, CM-TAG deposition as tissue lipid was preserved, despite decreased CM-TAG oxidation; metabolic fate of VLDL-TAG was unaffected by NEFA. TAG (CM or VLDL) in the perfusate tended to decrease lipoprotein lipase (LPL) activity; this may be a reflection of increased LPL turnover in the presence of lipoproteins.     

Hepatic actions of levonorgestrel: correlations between biochemical and morphological findings

The influence of the synthetic sexual steroid levonorgestrel (LN) on rat liver in various doses and at different structural levels was investigated. A slight reactive hepatosis was found by histological examination after administration of LN in a dose of 10 mg per kg body wt. The same dose caused exclusively distinct lesions of the mitochondria, however, only in centrilobular parenchymal cells, whereas in the periportal hepatocytes only the lipid droplet content appears somewhat elevated. LN decreased the total glutathione content of the liver. The mitochondrial glutathione was decreased more intensively. One mg/kg body wt. of LN decreased the cytochrome P-450 content, but 10 mg/kg body wt. increased ethyl-morphine N-demethylation and 7-ethoxycoumarin O-deethylation activities. Distinct correlations could be shown between the biochemical changes and the ultrastructural findings.     

Stabilized ferrous gluconate as iron source for food fortification: bioavailability and toxicity studies in rats

The iron bioavailability and acute oral toxicity in rats of a ferrous gluconate compound stabilized with glycine (SFG), designed for food fortification, was studied in this work by means of the prophylactic method and the Wilcoxon method, respectively. For the former studies, SFG was homogeneously added to a basal diet of low iron content, reaching a final iron concentration of 20.1 +/- 2.4 mg Fe/kg diet. A reference standard diet using ferrous sulfate as an iron-fortifying source (19.0 +/- 2.1 mg Fe/kg diet) and a control diet without iron additions (9.3 +/- 1.4 mg Fe/kg diet) were prepared in the laboratory in a similar way. These diets were administered to three different groups of weaning rats during 23 d as the only type of solid nourishment. The iron bioavailability of SFG was calculated as the relationship between the mass of iron incorporated into hemoglobin during the treatment and the total iron intake per animal. This parameter resulted in 36.6 +/- 6.2% for SFG, whereas a value of 35.4 +/- 8.0% was obtained for ferrous sulfate. The acute toxicological studies were performed in two groups of 70 female and 70 male Sprague-Dawley rats that were administered increasing doses of iron from SFG. The LD50 values of 1775 and 1831 mg SFG/kg body wt were obtained for female and male rats, respectively, evidencing that SFG can be considered as a safe compound from a toxicological point of view.     

Effects of angiotensin II, arginine-vasopressin, endothelin and catecholamines on plasma atrial natriuretic peptide concentrations in the conscious newborn calf.

The effects of epinephrine (E), norepinephrine (NE), angiotensin II (AII), arginine-vasopressin (AVP) and endothelin on plasma ANP levels were studied according to a latin square design in six 12-21 days-old conscious Jersey calves weighing 30 +/- 4 kg. The animals chronically-instrumented with a carotid catheter for blood pressure recording, received at 11.00 a.m. an i.v. right jugular continuous infusion for 30 min of two different sub-pressor or pressor dose-levels of each substance; E: 0.6 and 5.5 nmol/min per kg body wt; NE: 0.6 and 6 nmol/min per kg body wt; AII: 9.6 and 96 pmol/min per kg body wt; AVP: 0.6 and 69 pmol/min per kg body wt; and endothelin: 1.2 and 12 pmol/min per kg body wt). Control animals received, in the same way, the same volume (2 ml/kg body wt) of NaCl 0.9%. In Jersey calves, basal plasma atrial naturetic peptide (ANP) levels were around 5 pmol/l. Marked increases in this parameter were produced by all substances when given at the highest dose-level. The maximal rise of 600% was observed with AII; however on a molar basis, endothelin appeared more potent than AII and at the same dose-level, E appeared more effective than NE to increase circulating ANP (17.8 +/- 0.3 vs 9.5 +/- 0.1 respectively at time 70 min; P less than 0.01). The time-course of plasma ANP levels was positively correlated (P less than 0.01) by linear regression with the increase in blood pressure when pressor agents were given at the highest dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of corticosteroid on the transport and metabolism of glutamine in rat skeletal muscle.

Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na+, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na+ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 +/- 75 and 60 +/- 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (Vmax) for glutamine transport into muscle (by Na(+)-glutamine symport) fell from 1058 +/- 310 to 395 +/- 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the Km (from 8.1 +/- 1.9 to 2.1 +/- 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in Vmax and Km of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na+ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine.     

Purification and characterization of glutathione disulfide-stimulated Mg2+-ATPase from human erythrocytes

We have previously shown the presence of two different forms of glutathione disulfide (GSSG)-stimulated Mg2+-ATPases in human erythrocytes. We have now investigated a low-Km form of the enzyme from human erythrocytes. Purification of the enzyme was performed to apparent homogeneity involving procedures of affinity chromatography and gel filtration. The enzyme was composed of two non-identical subunits of Mr = 82K and 62K. The enzyme reconstituted into phospholipid vesicles showed both GSSG-stimulated Mg2+-ATPase activity (285 nmol Pi released/mg protein/min) and active GSSG transport activity (320 nmol GSSG/mg protein/min). The amino acid composition of the enzyme was similar to that of the enzyme purified from cytoplasmic membranes of human hepatocytes. These enzymes were immunologically cross reactive. These results indicate that this enzyme functions in the active transport of GSSG as it possibly does in hepatocytes.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by calcium in rat hepatocytes

The effects of Ca2+, ionophore A23187, and vasopressin on CTP:phosphocholine cytidylyltransferase were investigated. Cytidylyltransferase is present in the cytosol and in a membrane-bound form on the microsomes. Digitonin treatment caused release of the cytosolic form rapidly. Addition of 7 mM Ca2+ to hepatocyte medium resulted in a 3-fold decrease in cytidylyltransferase released by digitonin treatment (1.7 +/- 0.1 nmol/min per mg compared to 5.1 +/- 0.2 nmol/min per mg in the control). Verapamil, a calcium channel blocker, partially overcame this effect of Ca2+. Ionophore A23187 and vasopressin both mimicked the effect of Ca2+ and resulted in a decrease in cytidylyltransferase release (2.4 +/- 0.1 nmol/min per mg and 2.5 +/- 0.2 nmol/min per mg, respectively) compared to control (3.4 +/- 0.1 nmol/min per mg). In agreement with the digitonin experiments, incubation with 7 mM Ca2+ resulted in a decrease in cytidylyltransferase in the cytosol (from 4.0 to 1.2 mol/min per mg) and a corresponding increase in the microsomes (from 0.6 to 2.4 nmol/min per mg). Verapamil partially blocked this translocation caused by Ca2+. Ionophore A23187 and vasopressin also caused translocation of the cytidylyltransferase from the cytosol to the microsomes. The addition of Ca2+ also resulted in an increase in PC synthesis. With 7 mM Ca2+ in the medium, the label associated with PC increased to 3.8 +/- 0.1.10(6) dpm/dish from 2.7 +/- 0.1.10(6) dpm/dish after 10 min. PC degradation was also affected, since 7 mM Ca2+ in the medium resulted in an increase in LPC formation both in the cell and the medium. We conclude that high concentrations of calcium in the hepatocyte medium can cause a stimulation of CTP:phosphocholine cytidylyltransferase and PC synthesis in cultured hepatocytes.