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T Noll

Publications and source records attributed to T Noll.

At least 19 recordsLinked to original sources
PubMed1992

Mitochondrial ATP-synthase activity in cardiomyocytes after aerobic-anaerobic metabolic transition.

It has been hypothesized that, in oxygen-depleted myocardial cells, mitochondria are depolarized and the F1,F0-proton adenosinetriphosphatase (ATPase) catalyzes net ATP hydrolysis when the cells exhibit the signs of an aerobic-anaerobic metabolic transition, which are increased lactate formation and decline in high-energy phosphate reserves [W. Rouslin, C. W. Broge, and I. L. Grupp. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1759-H1766, 1990]. This hypothesis was tested by incubating isolated cardiomyocytes from the adult rat in substrate-free Tyrode solution (37 degrees C, pH 7.4) at a PO2 less than or equal to 0.1 Torr, i.e., 1,000-fold below the normal arterial level. At this deep hypoxia, the following results were found. 1) Lactate production was activated to maximal rates and high-energy phosphate contents decreased (aerobic-anaerobic metabolic transition). The inhibitor of the mitochondrial F1,F0-proton ATPase oligomycin, however, added upon establishment of hypoxia, did not slow down, as in the case of depolarized mitochondria, but moderately accelerated energy depletion. 2) Activation of mitochondrial ATP hydrolysis could be provoked in these hypoxic cells by addition of cyanide, antimycin A, and rotenone, i.e., specific inhibitors of certain sites of the respiratory chain. The enhancement of loss of ATP could be inhibited by oligomycin. The results demonstrate that states of deep hypoxia of the cardiomyocyte are possible in which it undergoes an aerobic-anaerobic metabolic transition, indicated by increased lactate formation and progressive loss of cellular energy reserves, and yet mitochondrial ATPase hydrolytic activity is not activated.

2,4-Dinitrophenol↗
PubMed1992

Cytosolic Ca2+ overload and macromolecule permeability of endothelial monolayers.

It was investigated how cytosolic Ca2+ overload affects the cytoskeletal structure and macromolecule permeability (for albumin) of monolayers of endothelial cells (from porcine aorta). States of cytosolic Ca2+ overload were produced either 1. by metabolic inhibition (5 mM KCN plus 5 mM 2-deoxyglucose) or 2. by increasing membrane permeability with the use of a Ca2+ ionophore (10 microM A 23187). The effects of cytosolic Ca2+ overload on the structure of F-actin filaments and monolayer permeability were monitored. ATP stores were rapidly degraded (> 90% in 15 minutes) in the presence of metabolic inhibitors, but only partially reduced in the presence of A 23187 (30%) in two hours). Concomitantly with ATP loss, cytosolic Ca2+ levels were increased in metabolically inhibited cells. Two-hour exposure to the Ca2+ ionophore A 23187 mimicked the effect of two-hour metabolic inhibition on F-actin filaments and monolayer permeability, in spite of the divergence in energy metabolism. Disintegration of F-actin filaments in presence of metabolic blockers or ionophore was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. Within three hours after removal of these agents, a normal F-actin structure and normal macromolecule permeability were re-established in the monolayers. The results show that cytosolic Ca2+ overload causes disintegration of F-actin filaments and a subsequent increase in macromolecule permeability. These changes are readily reversible as long as the dis-integration is based on fragmentation and not depolymerization of F-actin filaments.

Actins↗
PubMed1990

Energetic response of coronary endothelial cells to hypoxia.

The response of endothelial energy metabolism to oxygen supply was studied in cultured coronary endothelial cells from the rat at defined PO2 levels between 0.1 and 100 Torr. In the presence of glucose (5 mM), endothelial respiration (4 nmol O2.min-1.mg protein-1) was independent of the exterior PO2 greater than 3 Torr; oxygen consumption was half maximal at 0.8 Torr. At 100 Torr, lactate production was 26 nmol.min-1.mg protein-1; the decrease of the PO2 to 0.1 Torr resulted in a 2.2-fold increase in lactate production. The contents of ATP, ADP, and AMP were 21, 4, and 2 nmol/mg protein, respectively; they remained constant for 2.5-h incubations at PO2 levels between 0.1 and 100 Torr. In the presence of palmitate (100 microM) plus glutamine (0.5 mM), oxygen consumption was 8 nmol.min-1.mg protein-1 at PO2 levels greater than 3 Torr, and the half-maximal rate was again observed at 0.8 Torr. Lactate production was negligible. At PO2 levels greater than 3 Torr, the cells remained well energized. Below 3 Torr, however, the adenine nucleotide contents rapidly declined. These results demonstrate that the oxygen demand of coronary endothelial cells is low compared with the beating myocardium. In the presence of glucose, aerobic glycolysis is pronounced and the Pasteur effect small. In severe hypoxia (PO2 less than 0.1 Torr) the energetic state remained stable. In the absence of glucose, the energetic state of coronary endothelial cells is sensitive to the exterior PO2 less than 3 Torr, declining concomitantly with the decrease in respiration.

Animals↗
PubMed1989

Halomethane hepatotoxicity: induction of lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under low oxygen partial pressures.

Halomethane-induced lipid peroxidation and inactivation of cytochrome P-450 were studied in liver microsomes from phenobarbital-pretreated rats in the presence of NADPH at steady-state O2 partial pressures (PO2). As indicated by the formation of thiobarbituric acid-reactive material and the stimulation of O2 uptake, significant lipid peroxidation was induced by those halomethanes containing more than two Cl, Br, or I atoms. Lipid peroxidation decisively depended on the PO2 present, showing distinct maxima at PO2 between 1 and 10 mm Hg. Those halomethanes inducing lipid peroxidation also led to inactivation of microsomal cytochrome P-450, as indicated by a loss of cytochrome P-450 detectable as ferrous CO complex and an equimolar loss of microsomal heme. Under anaerobic conditions inactivation of cytochrome P-450 presumably resulted solely from an attack of halomethane radicals on its heme moiety. Under aerobic conditions lipid peroxidation made an additional contribution to the inactivation of cytochrome P-450. These results suggest that the reductive activation to free radicals, catalyzed by cytochrome P-450, and thus the induction of lipid peroxidation at low but physiological PO2 are characteristic not only of CCl4 but also of other polyhalogenated methanes, especially CBrCl3, CBr4, CHI3, CHBr3, and CHBr2Cl.

Animals↗
PubMed1988

Lipid peroxidation and cell viability in isolated hepatocytes in a redesigned oxystat system: evaluation of the hypothesis that lipid peroxidation, preferentially induced at low oxygen partial pressures, is decisive for CCl4 liver cell injury.

An oxystat system is described which is capable of maintaining steady-state oxygen partial pressures (PO2) at levels between 0.1 and 300 mm Hg for hours or even days in incubations of respiring cells. The system was used to study effects of the hepatotoxin carbon tetrachloride (CCl4) on lipid peroxidation and cell viability in isolated hepatocytes from phenobarbital-pretreated rats at various steady-state PO2. At PO2 below 35 mm Hg, with a maximum effect at 7 mm Hg, CCl4 induced an immediate lipid peroxidation, the rate of which slowed down during further incubation. AT PO2 between 35 and 70 mm Hg, CCl4 initially induced only slight lipid peroxidation, while there was a significant increase in lipid peroxidation after approximately 30 min. At PO2 above 100 mm Hg, no lipid peroxidation was induced by CCl4. At PO2 of 70 mm Hg and below, with the maximum effect at 3 mm Hg, CCl4 also induced marked losses of cell viability. Under anaerobic conditions and at PO2 greater than 70 mm Hg, CCl4 was without effect on the viability of the liver cells. Cells isolated from the pericentral area of the liver lobule showed more lipid peroxidation and loss of cell viability than cells from the periportal area of the lobule. These results provide further evidence for the decisive role of lipid peroxidation, preferentially induced at low PO2, in CCl4 liver injury.

Animals↗
PubMed1988

Dependence of mitochondrial and cytosolic adenine nucleotides on oxygen partial pressure in isolated hepatocytes. Application of a new rapid high pressure filtration technique for fractionation.

By using a new rapid high pressure filtration technique, mitochondrial and cytosolic ATP and ADP contents were determined in isolated hepatocytes at different oxygen partial pressures. At 670 mmHg, subcellular adenine nucleotide contents and ATP/ADP ratios were comparable with values obtained with the digitonin fractionation technique. However at lower oxygen partial pressure ADP appears to be rephosphorylated during digitonin fractionation whereas with high pressure filtration fractionation rephosphorylation of ADP is avoided due to shorter fractionation times. Cytosolic and mitochondrial ATP/ADP ratios decrease if oxygen partial pressure is lowered. However the absolute values of ATP/ADP ratios depend critically on the incubation conditions. Thus incubation of hepatocytes in an oxystat system, where oxygen partial pressure is maintained constant by infusing oxygen-saturated medium and the hepatocyte suspension is continuously stirred, yields much higher subcellular and overall ATP/ADP ratios than incubation in Erlenmeyer flasks gassed with different gas mixtures and shaken in a water bath. This is ascribed to limited diffusion of oxygen from the medium into the cell if the suspension is not mixed thoroughly by stirring. The strong dependence of subcellular ATP/ADP ratios on incubation conditions indicates that oxygen may be one rate-controlling factor for oxidative phosphorylation in the intact cell.

Adenine Nucleotides↗
PubMed1987

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation.

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.

Adenosine Diphosphate↗
PubMed1987

Dependence of hepatic gluconeogenesis on PO2: inhibitory effects of halothane.

The dependence of gluconeogenesis and O2 uptake on PO2 in isolated rat hepatocytes is presented. Maintenance of steady-state PO2 was achieved with an oxystat system (Biochem. J. 236: 765-769, 1986). O2 uptake showed a half-maximal (K0.5) value of 0.5 Torr PO2, whereas the glucose synthesis rate was half-maximal at 1.2 Torr PO2. Halothane at concentrations greater than 1 mM exerted a parallel inhibition of O2 uptake and glucose synthesis at all PO2 levels studied. In contrast, at halothane concentrations less than 1 mM, inhibition of glucose synthesis occurred only at less than 20 Torr PO2. At these low concentrations, halothane was without significant effects on cellular O2 uptake. In isolated mitochondria, inhibition of O2 uptake was already half-maximal at a halothane concentration of 0.5 mM. In this subcellular system the inhibitory effect of halothane was independent of PO2. These results demonstrate that the critical PO2 at which cellular O2 utilization begins to decrease and the PO2 at which glucose synthesis begins to decrease are comparable; both PO2 levels are approximately 5 Torr. The metabolic zonation of the liver lobule is discussed in view of the results presented.

Animals↗
PubMed1987

The decisive pO2-levels in haloalkane-mediated liver cell injury.

The model hepatotoxin carbon tetrachloride (CCl4) was used to study haloalkane free radical-induced lipid peroxidation in isolated rat hepatocytes at steady state oxygen partial pressures (pO2) between 0.2 and 100 mmHg. Equilibrium oxygen conditions were achieved by using an oxystat system. Monitoring of hepatocellular oxygen uptake, malondialdehyde-formation and low-level chemiluminescence during incubations of CCl4-supplemented hepatocytes indicated a drastic stimulation of lipid peroxidation at pO2-levels between 1 and 10 mmHg. Above and below this pO2-region the potency of CCl4 to induce lipid peroxidation sharply decreased. The evaluation of cellular damages by determining trypan blue exclusion and lactate dehydrogenase leakage revealed that in the presence of CCl4 hepatocellular injury was significantly increased at those pO2-levels which were optimal for CCl4-mediated lipid peroxidation. The present results demonstrate that CCl4 is a potent inducer of lipid peroxidation also in the intact hepatocyte, provided that the pO2 is maintained at distinct low levels. The coincidence of lipid peroxidation and loss of cell viability at the same pO2-range provides further evidence for the assumption that the haloalkane-mediated liver cell injury is due to a peroxidative process which primarily occurs at the hypoxic end of the physiological pO2-levels (1-70 mmHg) in liver.

Animals↗
PubMed1986

A computer-supported oxystat system maintaining steady-state O2 partial pressures and simultaneously monitoring O2 uptake in biological systems.

A feedback-controlled oxystat system is described maintaining steady-state O2 partial pressures (pO2) between 0.01 mmHg (14 nM-O2) and 150 mmHg (210 microM-O2) and simultaneously monitoring O2 uptake at rates between 0.1 and 120 microM-O2 X min-1 in suspensions of cells, in subcellular fractions and in solutions of enzymes. At pO2 values between 0.2 and 150 mmHg (0.28 and 210 microM-O2) a polarographic O2 sensor was used, and below a pO2 of 0.2 mmHg (0.28 microM-O2) the O2-dependent luminescence of the photobacterium Vibrio fischeri was utilized to monitor the actual pO2. At a selected pO2, O2 supply is maintained by injecting appropriate amounts of O2-saturated aqueous medium into the reaction chamber by using a motor-driven burette. The oxystat system is under control of a computer that reads the O2 sensors, interacts with the motor-driven burette, calculates the O2 uptake from the amounts of O2-saturated medium added, collects data from further measuring devices and provides the documentation of the results during incubation.

Computers↗
PubMed1986

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation.

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.

Animals↗
PubMed1985

Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.

5'-Nucleotidase↗
PubMed1985

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase.

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.

Acid Anhydride Hydrolases↗
PubMed1985

Oxygen dependence and subcellular partitioning of hepatic menadione-mediated oxygen uptake. Studies with isolated hepatocytes, mitochondria, and microsomes from rat liver in an oxystat system.

Using an oxystat system, menadione (2-methyl-1,4-naphthoquinone)-mediated oxygen uptake was investigated in isolated rat hepatocytes, in malate/glutamate-supplemented mitochondria, and in NADPH-reduced microsomes at steady-state oxygen partial pressures (pO2) between 0.1 to 100 mm Hg (0.2-150 microM O2). Menadione-mediated stimulation of oxygen uptake was half-maximal at pO2 of 0.5, 0.2, and 0.9 mm Hg, respectively. In hepatocytes and mitochondria half-maximal concentrations of menadione were 15 and 4 microM. However, in microsomes saturation with menadione was not reached at concentrations up to 300 microM. Antimycin A inhibited menadione-mediated oxygen uptake in hepatocytes and mitochondria by about three-fourths, while rotenone was without inhibitory effect; KCN inhibited practically completely. In mitochondria menadione-stimulated oxygen uptake was significantly inhibited by dicoumarol but further enhanced by the addition of ADP, even in the presence of rotenone. The results suggest that menadione-mediated hepatocellular oxygen uptake proceeds almost independently of pO2 in most regions of the liver lobule but that in areas of low pO2 such as the centrolobular regions limitation by oxygen may occur. They also demonstrate that in the intact hepatocyte menadione-mediated oxygen uptake predominantly (greater than 90%) results from electron transfer in the mitochondrial respiratory chain by menadione.

Animals↗
PubMed1985

Halothane-induced lipid peroxidation and glucose-6-phosphatase inactivation in microsomes under hypoxic conditions.

Halothane-induced lipid peroxidation was studied in microsomes from phenobarbital-pretreated male rats at defined steady state oxygen partial pressures (PO2). At PO2 less than 10 mmHg on addition of halothane to NADPH-reduced microsomes, significant increases in malondialdehyde (MDA) formation, oxygen uptake, and conjugated dienes were measured. At the maximum, near a PO2 of 1 mmHg, halothane induced the formation of about 0.75 nmol MDA X mg microsomal protein-1 X min-1; it also stimulated microsomal oxygen uptake twofold to threefold, and caused an almost threefold increase in conjugated diene absorption. Moreover, at this PO2 microsomal glucose-6-phosphatase lost about 70% of its activity. At PO2 greater than 10 mmHg, no significant effects of halothane on MDA formation, oxygen uptake, conjugated diene absorption, and glucose-6-phosphatase activity were observed; likewise under anaerobic conditions there was only a slight increase in conjugated dienes. The findings demonstrate that halothane induces microsomal lipid peroxidation at low PO2 and in the presence of particular cytochrome P-450 isoenzymes, and that the halothane-induced lipid peroxidation leads to severe microsomal lesions, as indicated by the loss of glucose-6-phosphatase activity.

Animals↗