The regulation of hepatic glutathione.
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Animals↗
Biomedical subjects
T Y Aw
Publications and source records attributed to T Y Aw.
ATP concentration gradients in cytosol of liver cells during hypoxia.
The activities of two ATP-requiring systems with different subcellular localizations were studied in cells in which average cellular ATP concentration was varied. The cytosolic ATP-sulfurylase activity varied linearly with the cellular ATP concentration; however, the plasma membrane Na+-K+-ATPase was substantially more sensitive to decreased ATP concentration. Under conditions where the cellular ATP concentration was lowered to 40% of control, Rb+ uptake was nearly zero. The results indicate that ATP-utilizing enzymes located in the plasma membrane in liver cells are exposed to a lower ATP concentration than are enzymes present in the cytosolic fluid surrounding the mitochondria. Analysis of radial diffusion of ATP from mitochondria, assuming that mitochondria are spherical and ATP consumption is zero order in ATP concentration, shows that the average ATP supply radius decreases as mitochondrial ATP production decreases. Hence, during limited ATP supply, enzymes with a greater average distance from mitochondria (e.g., plasma membrane ATPases) experience a more dramatic decrease in ATP concentration than enzymes in close proximity to mitochondria (e.g., cytoplasmic enzymes). Thus microheterogeneity of ATP supply can occur in cells without membranal compartmentation due to nonuniform distribution of ATP-generating and ATP-consuming systems.
Adenosine Triphosphate↗
Sinusoidal efflux of glutathione in the perfused rat liver. Evidence for a carrier-mediated process.
Turnover of hepatic glutathione in vivo in the rat is almost entirely accounted for by cellular efflux, of which 80-90% is sinusoidal. Thus, sinusoidal efflux play a major quantitative role in homeostasis of hepatic glutathione. Som preliminary observations from our laboratory (1983. J. Pharmacol. Exp. Ther. 224:141-147.) and circumstantial evidence in the literature seemed to imply that the raising of the hepatic glutathione concentration above normal was not accompanied by a rise in the rate of sinusoidal efflux. Based on these observations, we hypothesized that the sinusoidal efflux was probably a saturable process and that at normal levels of hepatic glutathione the efflux behaved as a zero-order process (near-saturation). We tested our hypothesis by the use of isolated rat livers perfused in situ, single pass, with hemoglobin-free, oxygenated buffer medium at pH 7.4 and 37 degrees C. Preliminary experiments established a range of perfusion rates (3-4 ml/min per g) for adequacy of oxygenation, lack of cell injury, and minimization of variability contributed by perfusion rates. Hepatic glutathione was lowered to below normal by a 48-h fast, diethylmaleate (0.1-1.0 ml/kg i.p.), and buthionine sulfoximine (8 mmol/kg i.p.), and raised to above normal by 3-methylcholanthrene (20 mg/kg x 3 d i.p.) and cobalt chloride (0.05-0.27 g/kg-1 subcutaneously). Steady state sinusoidal efflux from each liver was measured over a 1-h perfusion, during which the coefficient of variation of glutathione in perfusates stayed within 10%. Hepatic glutathione efflux as a function of hepatic concentration was characterized by saturable kinetics with sigmoidal (non-hyperbolic) features. The data were fitted best with the Hill model and the following parameter values were estimated: Vmax = 20 nmol/min per g, Km = 3.2 mumol/g, and n = 3 binding/transport sites. The efflux could be inhibited reversibly by sulfobromophthalein-glutathione conjugate but was not affected by the addition of glutathione to the perfusion medium. The results support our hypothesis that sinusoidal efflux of glutathione is near saturation (approximately equal to 80% of Vmax) at normal (fed and fasted) liver glutathione concentrations. The phenomenon of saturability coupled with the ability to inhibit the efflux leads us to propose that sinusoidal efflux from hepatocytes appears to be a carrier-mediated process. Some recent studies by others, using sinusoidal membrane-enriched vesicles, also support these conclusions.
Animals↗
Inhibition of glutathione efflux from isolated rat hepatocytes by methionine.
A substantial inhibition (50-70%) of GSH efflux by methionine was demonstrated in hepatocytes isolated from fed rats. Concurrent measurements of intracellular GSH revealed maintenance of a higher concentration in methionine-supplemented cells over the 1-h incubation. Analysis of total GSH suggested that maintenance of higher intracellular GSH by methionine could be quantitatively accounted for by inhibition of GSH efflux rather than by net GSH synthesis. This conclusion was supported by studies with propargylglycine, a potent inhibitor of cysteine synthesis from methionine. Identical results were obtained in incubations containing either propargylglycine and methionine or methionine alone, thereby suggesting that net synthesis of GSH from methionine was minimal under the assay conditions. Similar decreases (40-60%) in the rate of extracellular accumulation of GSH were observed with ethionine and buthionine, two higher homologs of methionine, but not with a wide range of other naturally occurring and synthetic amino acids. The inhibition of GSH efflux by methionine was not dependent on the presence of sodium in the medium and did not correlate with metabolic consumption of ATP.
Alkynes↗
Control of glucuronidation during hypoxia. Limitation by UDP-glucose pyrophosphorylase.
The regulation of glucuronidation during hypoxia was studied in isolated hepatocytes by analysing the dependence of acetaminophen glucuronidation rate on the intracellular concentrations of UTP, glucose 1-phosphate, UDP-glucose and UDP-glucuronic acid. The steady-state concentrations of these metabolites in cells from fed and starved rats were altered by exposure to various hypoxic O2 concentrations and by adding exogenous glucose. Changes in glucuronidation rate under all conditions were explained in terms of the concentrations of the substrates for UDP-glucose pyrophosphorylase, i.e. UTP and glucose 1-phosphate. Steady-state rates for the UDP-glucose pyrophosphorylase reaction, calculated by using published kinetic constants and measured glucose 1-phosphate and UTP concentrations, were in agreement with the measured glucuronidation rates. Thus the UDP-glucose pyrophosphorylase reaction is the key regulatory site for drug glucuronidation during hypoxia. Control at this site indicates that glucuronidation in vivo may be generally depressed in pathological conditions involving hypoxia and energy (calorie) malnutrition.
Acetaminophen↗
Oxygen dependence of adrenal cortex cholesterol side chain cleavage. Implications in the rate-limiting steps in steroidogenesis.
The oxygen dependence of cholesterol side chain cleavage to form pregnenolone was measured, using both purified phospholipid vesicle-reconstituted cytochrome P-450scc and rat adrenal mitochondria. At saturating cholesterol and nonlimiting electron supply (via NADPH-adrenodoxin reductase and adrenodoxin) the Km(O2) is low (4 microM). Limitations in the availability of both cholesterol and reductant caused elevations in the observed Km(O2). Pregnenolone synthesis was measured in mitochondria from variously pretreated rats, using a phospholipid-cholesterol dispersion as the source of exogenous substrate. In mitochondria obtained from ether-stressed rats (which elevates adrenocorticotropic hormone) two phases of malate-supported pregnenolone production are seen, a rapid (first 2 min) highly oxygen-dependent phase (Km = 150 microM) and a slow (2-10 min) relatively oxygen-independent phase (Km less than 10 microM). Comparison of side chain cleavage rates with mitochondrial 11 beta-hydroxylation rates at various oxygen concentrations suggests that the rapid phase is limited by the availability of reducing equivalents. In cycloheximide-pretreated ether-stressed rats, only a linear slow rate of pregnenolone production was seen (about 25% of the rate of the slow phase in the ether-stressed group), while in mitoplasts from both groups only a linear rapid rate was seen. Data are consistent with the proposal (Privalle, C. T., Crivello, J. F., and Jefcoate, C. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 702-706) that the adrenocorticotropic hormone-regulated cycloheximide-inhibitable rate of cholesterol side chain cleavage is limited by the rate of cholesterol transfer from outer to inner mitochondrial membranes.
Adrenal Cortex↗
Intracellular inhibition of UDP-glucose dehydrogenase during ethanol oxidation.
The enzymatic basis for inhibition of drug glucuronidation during ethanol oxidation was investigated in isolated rat hepatocytes. The intracellular rate of glucuronidation was varied independently by controlling the steady-state O2 concentration and the concentrations of UDP-glucose and UDP-glucuronic acid were measured in the absence and presence of 20 mM ethanol. Ethanol caused substantial inhibition of the glucuronidation rate which corresponded to a significant decrease in UDP-glucuronic acid concentration but not in UDP-glucose concentration. A plot of glucuronidation rate as a function of cellular UDP-glucuronic acid concentration yielded a single curve for incubations with or without ethanol; a similar plot of glucuronidation against UDP-glucose concentration gave separate curves for the two incubation conditions. These results clearly define the UDP-glucose dehydrogenase reaction as the site of inhibition during ethanol oxidation.
Acetaminophen↗
Uptake of riboflavin by isolated rat liver cells.
The effect of riboflavin status on uptake was investigated in hepatocytes isolated from control, riboflavin-sufficient and riboflavin-deficient rats. The uptake exhibited biphasic characteristics with an initial rapid phase [13.2 +/- 1.8 pmol/(10(6) cells X minute)] for the first couple of minutes followed by a second slower phase which continued for over an hour. The accumulation of riboflavin at near equilibrium conditions was 2.5- and 5.2-fold greater than external concentration in control and riboflavin-deficient cells, respectively. An apparent Km of 12 +/- 1.3 microM and Vmax of 82.3 +/- 9.1 pmol/(10(6) cells X minute) were obtained for control and riboflavin-sufficient rats while a similar Km but higher Vmax were obtained with deficient animals. Correspondence of the Km to that of flavokinase for riboflavin suggested the possibility that uptake of the vitamin may occur via metabolic trapping, i.e., phosphorylation. As substantiation of this, the rate of uptake was decreased by lumiflavin and 2'-hydroxyethylflavin, which are competitive inhibitors, and by 7,8-dichloroflavin, a substrate for flavokinase. Furthermore, the uptake was found to be temperature-dependent and studies with carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) and ethionine indicated a requirement for ATP. These results showed that overall, entry of riboflavin into hepatocytes occurs predominantly by a facilitated diffusion process followed by rapid trapping by flavokinase-catalyzed phosphorylation to FMN.
Adenosine Triphosphate↗
Direct determination of UDP-glucuronic acid in cell extracts by high-performance liquid chromatography.
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Animals↗
Secondary bioenergetic hypoxia. Inhibition of sulfation and glucuronidation reactions in isolated hepatocytes at low O2 concentration.
The O2 dependence of cytochrome oxidase, cellular ATP/ADP ratio, and drug sulfation and glucuronidation were studied in isolated hepatocytes to examine secondary biochemical changes of hypoxia related to decreased cytochrome oxidase function. A decrease in cytochrome oxidase function as well as in cellular ATP/ADP ratio occurred at low O2 concentrations and their O2 dependencies gave p50 values of 3.2 and 2.8 microM O2, respectively. Changes in the ATP/ADP ratio corresponded directly with changes in the oxidation-reduction state of cytochrome oxidase. The p50 value for sulfation of acetaminophen (2.5 microM O2) corresponded with the values for ATP/ADP ratio and cytochrome oxidase. Addition of compounds that lowered cellular ATP/ADP ratio caused a corresponding inhibition of sulfation. Selective use of ethionine, an adenosyl-trapping agent, demonstrated that the O2 dependence of sulfation related directly to a decrease in the absolute ATP concentration. The p50 value for glucuronidation (5.2 microM O2) was higher than that for sulfation, but in spite of this difference, glucuronidation also correlated well with cellular ATP/ADP ratio when this ratio was decreased by either O2 limitation or metabolic inhibitors. These results demonstrate that the predominant limitation of drug sulfation and glucuronidation during hypoxia is due to decreased cytochrome oxidase function.
Adenosine Diphosphate↗
Digestion and absorption of milk triacylglycerols in 14-day-old suckling rats.
The fatty acid compositions of the major classes of lipid recovered from the contents of the stomach and small intestine, as well as, the chylomicron fraction of the serum of 14-day-old rats have been compared to that of the milk triacylglycerols. The results indicate that at least two lipolytic activities are involved. As well as pancreatic lipase in the small intestine, a second lipase is present in the stomach and removes approximately half of the medium-chain acids (8:0, 10:0, 12:0) which together comprise between 30 and 50% of the total milk fatty acids. Tracer amounts of radioactively labeled fatty acids were then fed with carrier milk lipid and the kinetics of the appearance of the label in the blood determined. The very rapid appearance (less than 5 minutes) of 8:0 and 10:0 suggested that these acids are absorbed directly across the stomach wall and transported as the free acid. The longer times required for the other acids fed indicate that they are abosrbed from the small intestine. While both 16:0 and 18:1 were predominantly esterified into triacylglycerols and secreted in the chylomicrons, 12:0 was transported mainly as the free acid via the portal vein. Unlabeled 9:0, 11:0 and 13:0 were each absorbed and transported as the next higher homolog.
Animals↗
Effect of intramolecular fatty acid distribution on aspects of triacylglycerol digestion and absorption studied in vitro.
The triacylglycerols from several natural sources have been used as substrates for in vitro assays of three different aspects of fat digestion and absorption. The triacylglycerol mixtures were chosen because they differed widely both in total fatty acid composition and in the intramolecular distribution of fatty acids. The assays were designed to test the rate of hydrolysis with pancreatic lipase, the ability to form insoluble calcium soaps during the hydrolysis, and the ability to form mixed micelles with bile during the hydrolysis. Differences in the rate of hydrolysis appeared to be related to the structure and the triacylglycerols from lard and human milk, both of which have palmitic acid esterified in the sn-2 position, were hydrolysed most rapidly. Similarly the ability to form calcium soaps was related to structure and those triacylglycerols which released saturated fatty acids on hydrolysis favoured calcium soap formation. While there was a range of abilities to form mixed micelles with bile no obvious correlation with structure was detected.
Bile↗
The pathophysiological significance of lipid peroxidation in oxidative cell injury.
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Animals↗
NF kappa b signaling in posthypoxic endothelial cells: relevance to E-selectin expression and neutrophil adhesion.
Our previous studies have implicated the nuclear transcription factor kappa B (NF kappa B) in the regulation of adhesion molecule expression in endothelial cells exposed to anoxia-reoxygenation (A/R) or a redox imbalance. The objectives of this study were (1) to define the kinetics of NF kappa B activation by examining I kappa B alpha degradation and the nuclear translocation of p65 in response to A/R or redox imbalance (induced by treatment of cells with diamide and buthionine sulfoximine) and (2) to determine whether the signal for I kappa B alpha degradation, nuclear translocation of p65, and E-selectin-mediated neutrophil adhesion is related to the activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTPase) and/or protein kinase C (PKC). The results demonstrate that both A/R and redox imbalance led to I kappa B alpha degradation within 30 min and the concomitant appearance of p65 in the nucleus, consistent with rapid cytosolic activation of NF kappa B and subsequent nuclear translocation of the activated p65 subunit. Inhibition of PKC blocked I kappa B alpha degradation and p65 translocation in A/R-challenged, but not redox-altered, endothelial cells. However, both A/R- and redox-induced NF kappa B activation was blocked by inhibition of PTK. Similarly, A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion were blocked by inhibition of PKC or PTK, while only PTK inhibited the redox-induced adhesion response. Pretreatment of cells with N-acetyl cysteine effectively blocked A/R- or redox-induced I kappa B degradation and significantly attenuated the respective neutrophil adhesion responses. Collectively, these findings indicate that A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion are mediated by both PKC and PTK, which signal rapid activation of NF kappa B. This A/R-induced NF kappa B signaling response appears to be mediated, at least in part, by intracellular redox imbalance.
Acetylcysteine↗