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PubMed1999

Effects of fructose and xylitol on the urinary excretion of adenosine, uridine, and purine bases.

To examine whether fructose and xylitol increase the plasma concentration and urinary excretion of adenosine, as well as uridine and purine bases (hypoxanthine, xanthine, and uric acid), we intravenously administered xylitol and, 2 weeks later, fructose, to five healthy subjects. Analyses of blood and urine samples obtained during these infusion studies demonstrated that fructose increased the urinary excretion of adenosine and uridine 11.9- and 105.5-fold, respectively, and caused only a small increase in the plasma concentrations of uridine and purine bases. It was further demonstrated that xylitol increased the urinary excretion of uridine 58.4-fold, with a marked increase in the plasma concentrations of purine bases and uridine but without an increase in the urinary excretion of adenosine. However, neither infusion increased the plasma concentration of adenosine. These results suggest that in addition to many organs, including the liver, fructose is significantly metabolized by an abrupt adenosine triphosphate (ATP) consumption in the kidney, leading to an increase in the urinary excretion of adenosine and uridine. They also suggest that xylitol is not significantly metabolized in the kidney.

Adenosine↗
PubMed2000

Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity.

We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.

Acrylic Resins↗
PubMed1998

High-energy phosphates in the frontal lobe correlate with Wisconsin Card Sort Test performance in controls, not in schizophrenics: a 31phosphorus magnetic resonance spectroscopic and neuropsychological investigation.

In recent years, a number of 31phosphorus magnetic resonance spectroscopy (P-MRS) studies on the frontal lobe of schizophrenics have been performed, reporting alterations of phospholipids and high-energy phosphates. Deicken et al. (1994b) recently found positive correlations between left frontal phosphomonoester% (PME%) levels and the performance of a specific frontal lobe task, the Wisconsin Card Sorting Test (WCST), in schizophrenics. In the present paper, the correlations between phospholipids and high-energy phosphates in the frontal lobe of 26 schizophrenics and 23 controls measured with a volume-selective P-MRS method were investigated. Overall, we could not find any correlations between WCST results and phospholipid levels, but in controls phosphocreatine% (PCr%) and PCr/adenenosine triphosphate (ATP) ratios were negatively correlated with test performance. Since PCr behaves as a buffer of ATP, in the sense that when ATP is consumed by neuronal activity PCr is catalysed rapidly to ATP, increased PCr% values and, moreover, increased PCr/ATP ratios point to a decreased ATP consumption. Thus, the correlations found between PCr% and PCr/ATP and test performance in controls point to an association between reduced performance in a specific frontal lobe task and decreased energy demanding processes at rest. This association was not found in schizophrenics, possibly due to the influence of neuroleptic medication or the disease process per se.

Adenosine Triphosphate↗
PubMed2003

Mechanistic appraisal of the effects of some protease inhibitors on ciliary beat frequency in a sequential cell culture system of human nasal epithelium.

The aim of this study was to investigate the suitability of a sequential monolayer-suspension culture system as a model to screen subacute effects of drug excipients on ciliary beat frequency (CBF). The CBF of the cultured cells was measured by computerized microscope photometry. Protease inhibitors (puromycin, bestatin, bacitracin, actinonin and thiomersal) were used as model compounds and the mechanisms of ciliary inhibition were investigated by probing the involvement of arachidonic acid metabolism, guanylate cyclase (cGMP), protein kinase C (PKC) and adenosinetriphosphate (ATP) inhibition. Bestatin concentration-dependently reduced CBF by inhibiting arachidonic acid metabolism, cGMP, PKC and endogenous ATP consumption. Thiomersal and DMSO used for dissolving actinonin reduced CBF (P<0.05) via a non-specific mechanism. Bacitracin (8 mM) and puromycin (135 mM) had no effect on CBF after acute exposure (15-30 min) (P>0.05), but significantly reduced the CBF by approximately 15.0% following daily 15-min exposure for 1 week. This study shows that (i) sequential monolayer-suspension culture system is a valid model to screen both acute and subacute effects of drug excipients on CBF; and (ii) bacitracin, puromycin and actinonin are more cilio-compatible than bestatin and thiomersal and as such are more potentially useful nasal absorption enhancer from ciliotoxicity perspective.

Cell Culture Techniques↗
PubMed1980

A novel enzymatic activity of phenylalanyl transfer ribonucleic acid synthetase from baker's yeast: zinc ion induced transfer ribonucleic acid independent hydrolysis of adenosine triphosphate.

Phenylalanyl-tRNA synthetase from baker's yeast in the presence of phenylalanine or other amino acids misactivated by the enzyme, ATP, and low concentrations of Zn2+ is able to hydrolyze ATP to AMP and PPi very efficiently. After dialysis of the enzyme against ethylenediaminetetraacetic acid (EDTA), this amino acid dependent but tRNAPhe-independent hydrolysis is suppressed to negligible levels. The ATP hydrolysis can be restored by the addition of Zn2+ to the EDTA-dialyzed enzyme. During aminoacylation of tRNAPhe the Zn2+-induced ATP hydrolysis parallels the aminoacylation reaction, leading to nonstoichiometric production of AMP. Mechanistically, we conclude that Zn2+ can be bound to phenylalanyl-tRNA synthetase and can influence the stability of ATP if an activatable amino acid is present. The influence of Zn2+, if any, on the aminoacylation of tRNAPhe is not known. In practice, this side reaction is of the utmost importance in all cases in which the fate of ATP during aminoacylation is followed, especially if the stoichiometry of ATP consumption in relation to Phe-tRNAPhe formation has to be determined.

Adenosine Triphosphate↗
PubMed2000

Role of high-energy phosphate metabolism in hydrogen peroxide-induced cardiac dysfunction.

This study was undertaken to clarify the role of high-energy phosphate metabolism in hydrogen peroxide-induced cardiac dysfunction using phosphorus and fluorine nuclear magnetic resonance spectroscopy. The exposure of a Langendorff-perfused heart to hydrogen peroxide (200-400 micromol/L, 8 min) provoked biphasic contractile dysfunction characterized by a transient depression of left ventricular developed pressure during the administration of hydrogen peroxide and a delayed elevation of left ventricular end-diastolic pressure after the washout of hydrogen peroxide. The initial phase of cardiac dysfunction correlated well with the accumulation of sugar phosphates (r = 0.89, p < 0.01). Furthermore, we demonstrated that glibenclamide, a potent inhibitor of the ATP-sensitive K+ channel, attenuated the initial depression of developed pressure. On the other hand, the delayed elevation of end-diastolic pressure correlated well with the total ATP depletion (r = 0.96, p < 0.01). However, ATP loss was supposed to be a mere result from the increased ATP consumption corresponding to a rise in intracellular free Ca2+ (from the control value of 315+/-23 nmol/L to 708+/-104 after the administration of hydrogen peroxide, p < 0.01), which also paralleled the elevation of end-diastolic pressure. Thus glycolytic inhibition and intracellular Ca2+ overload are independently responsible for the biphasic contractile dysfunction induced by hydrogen peroxide.

Adenosine Triphosphate↗
PubMed2001

Short-term effects of the 21-aminosteroid lazaroid tirilazad mesylate (PNU-74006F) and the pyrrolopyrimidine lazaroid PNU-101033E on energy metabolism of human peripheral blood mononuclear cells.

Two groups of antioxidant compounds, the 21-aminosteroids and the pyrrolopyrimidines, have been found to act as neuroprotective drugs against lipid peroxidation in the injured CNS. Like glucocorticoids at high doses they are assumed to produce their effects at least in part by direct membrane stabilizing effects. In order to prove this hypothesis, we have investigated in this study the effects of these drugs on the energy metabolism of activated human peripheral blood mononuclear cells (PBMC) since these cells have been shown to serve as a suitable test system for substances affecting processes of ATP turnover. We compared the in vitro effects of (i) the 21-aminosteroid lazaroid tirilazad, (ii) the pyrrolopyrimidine lazaroid PNU-101033E and (iii) the glucocorticoid methylprednisolone on mitogen-induced respiration rate and ATP-consumption. We show that tirilazad inhibits concanavalin A-stimulated respiration rate and sodium cycling across the plasma membrane. The effect of methylprednisolone is similar indicating corresponding cellular mechanisms. However, unlike methylprednisolone, tirilazad produced no significant effect on calcium cycling across the plasma membrane. PNU-101033E in our test system caused cytotoxic effects on PBMC that did not allow us to quantify cellular actions on energy metabolism. Our results underline the view that tirilazad, first, is mimicking the high-dose immunosuppressive pharmacology of glucocorticoids such as methylprednisolone and, second, is likely to produce its therapeutic effects by direct physicochemical interactions with cellular membranes.

Adenosine Triphosphate↗
PubMed2003

A mathematical model for the spontaneous contractions of the isolated uterine smooth muscle from patients receiving progestin treatment.

The in vitro spontaneous contractions of human myometrium samples can be described using a phenomenological model involving different cell states and adjustable parameters. In patients not receiving hormone treatment, the dynamic behavior could be described using a three-state model similar to the one we have already used to explain the oscillations of intrauterine pressure during parturition. However, the shape of the spontaneous contractions of myometrium from patients on progestin treatment was different, due to a two-step relaxation regime including a latched phase which cannot be simulated using the previous model without introducing an ad hoc mechanism to account for the extra energy involved in this sustained contraction. One way to do this is to introduce an anomalous rate of ATP consumption, the biochemical reasons for which have not yet been elucidated and which cannot be mathematically simulated using our experimental data. An alternative explanation is the reduced cycling rate of actin-myosin cross-bridges known to occur during the latch-phase. Our experimental findings suggest a third possibility, namely a sol-gel transition with a specific relaxation time constant, which would maintain a significant part of the cell population in the contracted-state until the intracellular-medium returns to its normal fluid behavior.

Adenosine Triphosphate↗
PubMed1990

Cyclosporin A and verapamil have different effects on energy metabolism in multidrug-resistant tumour cells.

Cyclosporin A (Sandimmune) rapidly induced an increase in daunorubicin accumulation in multidrug-resistant human ovarian carcinoma cells (2780AD) and was more potent than verapamil. Steady-state 3H-cyclosporin A accumulation at 37 degrees C in 2780AD cells was 60-70% of that in the sensitive A2780 cells. A rapid increase of ATP consumption and lactate production was induced in 2780AD cells by verapamil, but not by cyclosporin A. These results suggest that the interactions of cyclosporin A and verapamil with P-glycoprotein, which leads to inhibition of drug transport, have a different mechanistic basis.

Adenosine Monophosphate↗
PubMed1993

Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia.

During the first weeks of life, injury to the central nervous system caused by brief periods of oxygen deprivation greatly increases. To investigate possible causes for this change, the effects of hypoxia or application of the excitatory neurotransmitter glutamate on intracellular calcium ([Ca2+]i) and ATP were studied in rat cerebrocortical brain slices. [Ca2+]i was measured fluorometrically with the indicator Fura-2. Hypoxia (95% N2/5% CO2) or 100 microM sodium cyanide produced gradual elevations in [Ca2+]i and ATP depletion in slices from rats < 2 weeks old, but rapid changes in older rats. After 20 min, [Ca2+]i in adult slices exposed to cyanide was 1,980 +/- 310 nM; in day 1-14 animals, it was 796 +/- 181 nM (p < 0.05). Combination of cyanide and a glycolytic inhibitor (iodoacetate) rapidly elevated [Ca2+]i and depleted ATP in all age groups. Energy utilization during anoxia, assessed by measuring ATP fall in cyanide/iodoacetate-treated brain slices, increased with age. Elevations in [Ca2+]i caused by application of 500 microM glutamate increased 240% from days 1-2 to day 28, but ATP loss caused by glutamate did not change with age. The N-methyl-D-aspartate antagonist MK-801 delayed calcium entry during the initial 5-7 min of hypoxia or cyanide in rats < 2 weeks old. We conclude that anaerobic ATP production, conservation of energy by reduced ATP consumption, and reduced sensitivity to glutamate contribute to delaying elevation in [Ca2+]i in neonatal rat brain during hypoxia.

Adenosine Triphosphate↗
PubMed1973

Xylitol metabolism in the isolated perfused rat liver.

1. Loading the isolated perfused liver from well-fed rats with xylitol (20mm) caused a depletion of adenine nucleotides and P(i) and an accumulation of alpha-glycerophosphate. The ATP content fell to 66% of the control value after 10min and to 32% after 80min. The ADP and AMP contents also fell. After 80min 63% of the total adenine nucleotides and 59% of the P(i) had been lost. 2. The alpha-glycerophosphate content rose from 0.13 to 4.74mumol/g at 10min and reached 8.02mumol/g at 40min. 3. Xylitol was rapidly metabolized, the main products being glucose, lactate and pyruvate. 4. The [lactate]/[pyruvate] ratio in the presence of xylitol rose to 30-40. 5. On perfusion of livers from starved animals the main product of xylitol metabolism was glucose and the mean ratio xylitol removed/glucose formed was 1.29 (corrected for endogenous glucose and lactate production). This is close to the predicted value of 1.2. 6. Evidence is presented indicating that the loss of adenine nucleotides caused by xylitol is not due to the increased ATP consumption but to the accumulation of alpha-glycerophosphate and depletion of P(i). 7. The loss of adenine nucleotides accounts for the hyperuricaemia which can occur after xylitol infusion in man. 8. The relevance of the findings to the clinical use of xylitol as an energy source is discussed.

Journal Article↗
PubMed1981

Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium.

1. The contribution of Co2 fixation to the anaplerotic mechanisms in the myocardium was investigated in isolated perfused rat hearts. 2. K+-induced arrest of the heart was used to elicit a transition in the concentrations of the intermediates of the tricarboxylic acid cycle. 3. Incorporation of 14C from [14]bicarbonate into tricarboxylic acid-cycle intermediates was measured and the rates of the reactions of the cycle were estimated by means of a linear optimization program which solves the differential equations describing a simulation model of the tricarboxylic acid cycle and related reactions. 4. The results showed that the rate of CO2 fixation is dependent on the metabolic state of the myocardium. Upon a sudden diminution of cellular ATP consumption, the pool size of the tricarboxylic acid-cycle metabolites increased and the rate of label incorporation from [14C]bicarbonate into the cycle metabolites increased simultaneously. The computer model was necessary to separate the rapid equilibration between bicarbonate and some metabolites from the potentially anaplerotic reactions. The main route of anaplerosis during metabolite accumulation was through malate + oxaloacetate. Under steady-state conditions there was a constant net outward flow from the tricarboxylic acid cycle via the malate + oxaloacetate pool, with a concomitant anaplerotic flow from metabolites forming succinyl-CoA (3-carboxypropionyl-CoA).

Animals↗
PubMed2003

Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial proteins and by an intensification of the parallel activation of ATP usage and ATP supply (increase in direct stimulation of oxidative phosphorylation complexes accompanying stimulation of ATP consumption) during exercise.

Adaptation, Physiological↗
PubMed1982

Role of pyruvate carboxylation in the energy-linked regulation of pool sizes of tricarboxylic acid-cycle intermediates in the myocardium.

The increase in the metabolite pool size of the tricarboxylic acid cycle in the isolated perfused rat heart after a decrease in the ATP consumption by KCl-induced arrest was used to study the anaplerotic mechanisms. During net anaplerosis the label incorporation into the tricarboxylic acid-cycle intermediates from [1-14C]pyruvate increased and occurred mainly by pathways not involving prior release of the label to CO2. A method for determination of the specific radioactivity of mitochondrial pyruvate was devised, and the results corroborated the notion that tissue alanine can be used as an indicator of the specific radioactivity of intracellular pyruvate.

Alanine↗
PubMed1984

Isolation of cells from rabbit renal proximal tubules by using a hyperosmolar intracellular-like solution.

A novel method of isolation of cells from rabbit kidney proximal tubules by using an intracellular-like solution (ICS) and gentle mechanical agitation in the absence of enzymes or chelators is described. Metabolic and functional characteristics of these cells were studied after washing and resuspension in modified Hanks medium, and the results were compared with those obtained in cells similarly prepared in extra-cellular-like solution (ECS). Trypan Blue exclusion and protein content were not different between the two preparations. However, oxygen consumption, ATP content and time- and concentration-dependent rates of uptake of phosphate, alpha-methyl glucoside and L-alanine were severalfold higher in cells prepared in ICS. Na+-dependent uptake of these solutes was 95% and 80% of total uptake in cells prepared in ICS and ECS respectively. Maximum transport rates (Tmax.) of phosphate, alpha-methyl glucoside and L-alanine were significantly higher in cells prepared in ICS. We propose that the use of ICS in the isolation procedure would yield a functionally more viable cell preparation, and therefore provides an ideal model for transport and metabolic studies at a cellular level.

Alanine↗
PubMed1986

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.

Adenosine Triphosphate↗
PubMed1990

Regulation of platelet AMP deaminase activity in situ.

The regulation of platelet AMP deaminase activity by ATP, GTP and phosphate was studied in human platelets in situ, and in vitro after partial purification. In intact platelets, a similar 50% decrease in cytosolic ATP was induced by either glucose starvation or treatment with H2O2. During starvation, AMP deaminase was in the inhibited state, as ATP consumption was mostly balanced by the accumulation of AMP. During H2O2 treatment, however, the enzyme was in the stimulated state, as the AMP formed was almost completely deaminated to IMP. Cytosolic GTP fell by 40-50% in both starvation and H2O2 treatment. In contrast, intracellular phosphate was 4-5-fold higher in starved than in H2O2-treated cells. These data point to phosphate as the main regulator of AMP deaminase activity in situ. This conclusion was verified by kinetic analysis of partially purified AMP deaminase. At near-physiological concentrations of MgATP, MgGTP and phosphate, the S0.5 (substrate half-saturation constant) for AMP was 0.35 mM. Half-maximal stimulation by MgATP occurred at a concn. between 2 and 3 mM. This stimulation was antagonized by the inhibitory effects of phosphate (IC50 = 2.0 mM) and MgGTP (IC50 = 0.2-0.3 mM), which acted in synergism (IC50 is the concentration causing 50% inhibition). We conclude that the difference in adenylate catabolism between starved and H2O2-treated platelets is due to the distinct phosphate concentrations. During starvation, refeeding and H2O2 treatment, the values of the adenylate charge and the phosphorylation potential were kept closely co-ordinated, which may be effected by AMP deaminase.

AMP Deaminase↗
PubMed1997

Allosteric control of Zymomonas mobilis glucose-6-phosphate dehydrogenase by phosphoenolpyruvate.

The second enzyme of the Entner-Doudoroff glycolytic pathway in Zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (PEP). In the presence of PEP levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a Hill coefficient up to 2. At low ionic strength in the absence of PEP, the response to glucose 6-phosphate concentration is Michaelis-Menten, but at physiological ionic strength and pH, a Hill coefficient of 1.3 to 1.4 was found even in the absence of PEP. Km values for NAD+ and NADP+ are also ionic-strength-dependent, increasing rapidly as salt concentration increases. Some sigmoidicity was also observed for NAD+ in the presence of PEP at low glucose 6-phosphate concentrations. The results can be interpreted in a Monod-Wyman-Changeux model, in which glucose 6-phosphate binds principally to the R-state, PEP to the T-state, and NAD+ to both states. These observations are clearly physiologically significant, and provide an explanation for the control of the balance between glycolytic throughput and ATP consumption in Z. mobilis.

Allosteric Regulation↗