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B Quistorff

Publications and source records attributed to B Quistorff.

At least 73 records · Page 4Linked to original sources
PubMed1990

Hepatic zonation of insulin-stimulated tyrosine phosphorylation.

Zonal distribution of insulin stimulation of hepatic protein tyrosine phosphorylation, detected by immunoblotting with an anti-phosphotyrosine antibody, has been studied in the in situ perfused rat liver by dual-digitonin-pulse perfusion. Insulin promotes the rapid and sustained tyrosine phosphorylation of two proteins (pp150 and pp69) that are present only in the perivenous hepatocytes, while three others (pp46, pp48 and pp96) are stimulated identically in the periportal and perivenous cells. The ability of insulin to rapidly activate acetyl-CoA carboxylase is indistinguishable between the hepatic zones. Hepatic zonation of insulin-stimulated tyrosine phosphorylation could underly differential hepatic insulin responses and might provide clues to the identification of tyrosine phosphorylated proteins linked to insulin regulation of intracellular events.

Acetyl-CoA Carboxylase↗
PubMed1990

Early effects of radiotherapy in small cell lung cancer xenografts monitored by 31P magnetic resonance spectroscopy and biochemical analysis.

31P magnetic resonance spectroscopy (31P MRS) and biochemical analysis of extracts were applied to study the metabolic response to X-irradiation of small cell lung cancer in nude mice. Two small cell lung cancer xenografts, CPH SCCL 54A and 54B, with different radiosensitivity, although derived from the same patient, were studied. A total of 126 individual tumors were examined. Following 5.0-Gy irradiation, a reversible increase in the ATP/Pi ratio, reaching twice the pretreatment level within 2 wk, was observed with 31P MRS, while 20 Gy induced a reversible decrease in the ATP/Pi ratio. The t1/2 of this decline was 2 to 3 h for 54A and about 6 h for the less radiosensitive 54B. The 31P MRS data were compared with biochemical analysis of tumors freeze-clamped and extracted at similar intervals after 20 Gy. It appeared that an acute reversible increase in Pi concentration was the major cause of the ATP/Pi decrease induced by 20 Gy. A linear correlation between ATP/Pi estimated by 31P MRS and by analytical biochemistry was found. The ATP/Pi ratio may be valuable for early assessment of radiosensitivity of small cell lung cancer tumors.

Adenosine Triphosphate↗
PubMed1990

Phenobarbital protects cerebral cortex neurones against toxicity induced by kainate but not by other excitatory amino acids.

The effect of phenobarbital (PB) on the cytotoxicity induced by the excitatory amino acids (EAAs) glutamate, aspartate, N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was investigated in cultured cerebral cortex neurones. PB (100 microM) completely protected against the total cytotoxic damage induced by KA, whereas it had no such effect on the toxicity induced by any of the other EAAs. The IC50 for the inhibition of the KA induced toxicity was 56 microM. Furthermore, PB completely blunted the large increase in O2 uptake in the neurones seen during exposure of the cultures to KA. Since exposure of the cells to PB alone did not affect the basal O2 consumption in the cultures, these results suggest that PB directly interacts with the KA receptor or, alternatively, with processes which selectively are coupled to activation of the KA receptor but not to activation of other types of EAA receptors.

Amino Acids↗
PubMed1990

Inosine/pyruvate/phosphate medium but not adenosine/pyruvate/phosphate medium introduces millimolar amounts of 5-phosphoribosyl 1-pyrophosphate in human erythrocytes. A 31P-n.m.r. study.

Incubation of human erythrocytes in medium containing inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) at pH 6.6 leads to a more than 1000-fold increase in the concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP), as identified and quantified by 31P-n.m.r. spectroscopy. The accumulation is highly pH-dependent, with a maximum at extracellular pH 6.60, and the maximum value of 1.3-1.6 mmol/l of erythrocytes is attained within 1 h at 37 degrees C. PRPP was accumulated despite high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), an inhibitor of PRPP synthetase. The concentration of PRPP correlated with the intracellular concentration of inorganic phosphate (Pi). Substitution of either adenosine or adenosine plus inosine for inosine in the medium did not lead to 31P-n.m.r.-detectable accumulation of PRPP. These results show that neither 2,3-BPG nor PRPP itself inhibits the synthesis of PRPP in the human erythrocyte. Adenosine, however, prevents the inosine-stimulated accumulation of PRPP.

Adenosine↗
PubMed1990

Time resolved 3-dimensional recording of redox ratio during spreading depression in gerbil brain.

Optical fluorescence and reflectance measurements have been used to map the distribution of metabolic states in three dimensions in the gerbil brain with a spatial resolution of 200 microns an a time resolution of 4-6 s. In Mongolian gerbils anesthetized with pentobarbital, the redox states of the nicotinamide adenine dinucleotide (NADH) and flavoprotein components of the electron transport chain exhibit two distinct phases during the wave of spreading depression: (1) a transient period of oxidation and (2) a prolonged period of reduction, during which the cytochromes are reduced, and the hemoglobin is predominantly in the deoxy form. These data are interpreted as indicating that the energy demand placed on the gerbil brain during such spreading depression wave is sufficient to drive the brain temporarily hypoxic.

Animals↗
PubMed1990

A simple calf muscle ergometer for use in a standard whole-body MR scanner.

A calf muscle ergometer, designed for use in a standard whole-body MR scanner, is described. The instrument allows isotonic or isometric calf muscle exercise over a continuous range of work loads from 0 to individual maximum work output. Work output may be recorded on a strip chart recorder. A sound encoding provides an effective guidance for the subject tested to accurately reproduce his performance in each contraction cycle at a given work load. The FID recording may be triggered from the ergometer at any point during the contraction cycle. Nonmetal materials are used for the instrument construction in order to eliminate potentially disturbing eddy currents. The force resistance of the ergometer is obtained by a combined hydraulic-pneumatic system, which is loaded by coupling to an outside source of compressed air equipped with a suitable pressure regulator. When varying the time of data sampling within a repeated 6-s contraction cycle it was found that the recorded Pi/PCr ratio remained essentially constant at low and moderate work loads. However, at work loads at or above 70% of individual Vmax a pronounced dependence of the time of sampling with the contraction cycle was observed.

Equipment Design↗
PubMed1990

Ultrastructural changes of liver parenchyma following digitonin-pulse perfusion of rat liver.

It has been shown that pulse perfusion of rat liver with a digitonin-containing medium results in a highly zonated hepatocyte permeabilization, allowing selective sampling of cytosolic constituents from periportal and perivenous (centrolobular) hepatocytes "in situ". In the present paper we provide an ultrastructural evaluation of the perfusion method. Identical changes in hepatocytes from affected periportal and perivenous zones are found. Affected hepatocytes appear light (electron-lucent) in electron micrographs with a sharp transition to normal hepatocytes. The most conspicuous ultrastructural findings are: (1) transformation of the sinusoidal part of the light hepatocytes, the lipocyte processes and the endothelium of affected zones apparently unifying into a continuous layer dominated by disrupted plasma membranes and 7-nm filaments; (2) deposition of osmiophilic digitonin-cholesterol complexes along the sinusoidal plasma membranes of affected zones; and (3) reduction of the cytoplasmic matrix (cytosol) in the light hepatocytes, a dilation of the mitochondrial intermembrane space with a preserved mitochondrial matrix, and a dilation of cisternae of the granular endoplasmic reticulum. The ultrastructural findings are consistent with marker-enzyme activity measured in eluates from digitonin-perfused livers, except that lysosomes appear intact, apparently contrasting with the observed eluation of amyloglucosidase (Quistorff et al. 1985).

Animals↗
PubMed1990

Induction of millimolar amounts of 5-phosphoribosyl-1-pyrophosphate in human erythrocytes by incubation in inosine-pyruvate-phosphate medium. A 31P-NMR study.

Incubation of human erythrocytes in inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) (IPP-medium)1 at pH 6.6 leads to a ca. 1000 fold increase in the concentration of 5-phosphoribosyl-1-pyrophosphate (PRPP) as identified and measured by 31P-NMR spectroscopy. PRPP accumulation is pH-dependent with a maximum at extracellular pH 6.60 and the maximum value of 1.3-1.6 mmol/l RBC is reached within one hour at 37 degrees C. PRPP accumulated despite high concentrations of 2,3-diphosphoglycerate (2,3-DPG), an inhibitor of PRPP-synthetase. The concentration of PRPP attained correlated with the intracellular concentration of inorganic phosphate (P1). Substitution of either adenosine or adenosine plus inosine for inosine in the medium (APP or A+IPP media) did not lead to NMR detectable accumulation of PRPP, despite the same high concentrations of intracellular P1. The lack of accumulation of PRPP in adenosine containing media may be related to the higher concentrations of nucleoside diphosphates induced in these media. These results show that neither 2,3-DPG nor PRPP itself inhibit the synthesis of PRPP in the human erythrocyte under these circumstances.

Erythrocytes↗
PubMed1989

The Na+/K+-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study.

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.

Calibration↗
PubMed1989

A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study.

Metabolic control of oxidative metabolism was studied in perfused rat liver by means of phosphorus magnetic resonance spectroscopy. Oxygen consumption, ATP, and Pi were measured with different rates of gluconeogenesis and urea synthesis by varying concentrations of the substrates in the perfusate. Five levels of oxygen consumption (VO2) were obtained: an average control value of 1.94 +/- 0.14 and 2.93 +/- 0.25, 3.29 +/- 0.46, 3.85 +/- 0.26, and 4.18 +/- 0.56 mumol/min/g liver (mean +/- S.D., n = 6). The corresponding ATP concentrations were 2.51 +/- 0.20, 2.39 +/- 0.08, 2.24 +/- 0.09, 2.13 +/- 0.12, and 1.91 +/- 0.13 mM. Pi increased stoichiometrically with the decrease in ATP. Free Pi (Pif) was calculated as NMR-visible Pi in control plus -delta ATP (1.94 mM + (-delta ATP]. The kinetic relationship of oxidative phosphorylation as a function of Pif followed a Michaelis-Menten type of equation: VO2 = 5.55/(1 + 0.24/[( Pif] - 1.81]. The observed Km value for Pi of 0.24 mM approximates the reported Km value in isolated mitochondria of 1 mM. The free Pi concentration of 1.94 mM is in the range of the Km value, while the free ADP concentration of 200 microM exceeds the Km value of 20 microM. Therefore, it is suggested that Pi play a major role in the regulation of mitochondrial oxidative phosphorylation in combined urea synthesis and gluconeogenesis.

Adenosine Triphosphate↗
PubMed1989

Zonation of hepatic lipogenic enzymes identified by dual-digitonin-pulse perfusion.

The zonal distribution within rat liver of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase, the principal enzymes of fatty acid synthesis, was investigated by using dual-digitonin-pulse perfusion. Analysis of enzyme mass by immunoblotting revealed that, in normally feeding male rats, the periportal/perivenous ratio of acetyl-CoA carboxylase mass was 1.9. The periportal/perivenous ratio of ATP citrate-lyase mass was 1.4, and fatty acid synthase exhibited the largest periportal/perivenous mass gradient, having a ratio of 3.1. This pattern of enzyme distribution was observed in male rats only; in females, the periportal/perivenous ratio of enzyme mass was nearly equal. The periportal/perivenous gradients for acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthase observed in fed (and fasted) males were abolished when animals were fasted (48 h) and refed (30 h) with a high-carbohydrate/low-fat diet. As determined by enzyme assay of eluates obtained from the livers of normally feeding male rats, there is also periportal zonation of acetyl-CoA carboxylase activity, expressed either as units per mg of eluted protein or units per mg of acetyl-CoA carboxylase protein, suggesting the existence of gradients in both enzyme mass and specific activity. From these results, we conclude that the enzymes of fatty acid synthesis are zonated periportally in the liver of the normally feeding male rat.

ATP Citrate (pro-S)-Lyase↗
PubMed1989

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver. A re-interpretation of the microcirculatory zones.

The plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.

Animals↗
PubMed1989

Deuterium isotope effects on ethanol oxidation in perfused rat liver and in rats and rabbits in vivo: application to determine the contribution of various pathways.

The kinetic deuterium isotope effect, D(V/K), on ethanol oxidation was measured by the radiometric, competitive method using 14C-labelled ethanol containing deuterium in the (1-R) position. Acetate was isolated and used for the determination. Experiments were performed on rats either anaesthetized and laparotomized, or provided with indwelling catheters in a. carotis, v. cava and v. portae. Experiments were also made on perfused liver from rats pretreated with acetone, or a mixture of acetone and phenobarbital. Finally, intact non-anaesthetized rabbits were used. The apparent isotope effect in all in vivo experiments decreased rapidly in the presence of acetaldehyde as a consequence of the reversibility of the ADH reaction. In the case of rabbits and catheterized rats this problem was tackled by taking blood samples in quick succession, thus permitting extrapolation of the apparent isotope effect to the time of injection of the labelled ethanol. In anaesthetized rats injection of the ADH inhibitor isobutyramide was used to reduce the concentration of acetaldehyde and thereby the rate of decline of the apparent isotope effect. At high doses of isobutyramide the isotope effect was constant with time at about 1.9 suggesting the presence of non-ADH activity. In all three kinds of in vivo experiments the isotope effect ranged from 2.66 to 2.93. In the case of anaesthetized rats the mean value was 2.89 +/- 0.05 (S.D.). This figure is significantly different from that of rat liver ADH, P less than 0.001. As the figures for the initial isotope effects are minimum values the contribution of non-ADH ethanol oxidizing systems is likely to be small, probably less than 10 percent.

Acetaldehyde↗
PubMed1987

Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities.

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.

Alanine Transaminase↗
PubMed1986

Periportal and perivenous hepatocytes retain their zonal characteristics in primary culture.

Periportal and perivenous hepatocytes from rat liver were isolated by combined digitonin-collagenase perfusion, and gluconeogenesis, urea synthesis and fatty acid synthesis was measured both in freshly isolated cells and in primary culture. A periportal zonation of gluconeogenesis and urea synthesis of about 3 and 1.5 fold, respectively, was observed. This zonation persisted unchanged for 23 hours in culture under identical conditions of incubation for periportal and perivenous cells. Fatty acid synthesis was not zonated.

Alanine Transaminase↗