INFLUENCE OF POTASSIUM ON THE STEADY-STATE REDOX POTENTIAL OF THE ELECTRON TRANSPORT CHAIN IN SLICES OF RAT CEREBRAL CORTEX AND THE EFFECT OF OUABAIN

Abstract— The concentration dependence of the modifications by potassium of the respiratory intermediates in incubated slices of rat cerebral cortex has been examined in the presence and absence of calcium. In addition to the immediate increase in respiration and the concomitant oxidation of the respiratory intermediates, longer term increases in the steady-state redox potential were observed at higher potassium concentrations. Addition of calcium to the system did not appreciably alter the immediate effects of potassium, but shifted the redox state of the respiratory intermediates; these changes involved a decrease in reduced intermediate at low concentrations of potassium and a relatively higher level of reduced carriers at high concentrations of potassium. Ouabain (50 μ m ) inhibited both the initial responses to added potassium and modified the changes in steady-state levels of reduced intermediate in the absence of calcium. In the presence of calcium, ouabain (50 μ m ) inhibited the initial oxidation of NAD(P)H observed upon addition of potassium but had no effect on the respiratory response to the addition of low concentrations of potassium. The disassociation of these responses resulted in a large decrease in the steady-state levels of reduced cytochrome. At 30 m m potassium an oxidation of NAD(P)H was observed which accompanied by an increase in levels of reduced cytochromes. These changes in redox state of the respiratory carriers have been discussed in relation to previous reports dealing with the effects of potassium on aerobic glycolysis and oxygen consumption by brain slices.     

CYTOCHROME REDOX POTENTIAL DEPENDENCE ON SUBSTRATE IN RAT CEREBRAL CORTEX SLICES: IMPORTANCE OF CYTOPLASMIC NAD(P)H AND POTASSIUM

—Using dual-wavelength absorbance spectrophotometry, the ability of various substrates to produce and maintain a redox potential in the cytochrome chain of rat cerebral cortex slices was studied. In general, the ability to reduce the cytochromes parallels previously reported capabilities of the substrates to support metabolic responses to stimulation. The steady-state kinetics of cytochrome reduction by glucose or lactate displayed a very sharp dependency upon concentration in the regions of 1 or 3 mm , respectively. This was in contrast to a near linear reduction of the cytochromes with concentrations of pyruvate over a range of 1–10 mm . The production and maintenance of a cytochrome redox potential was found to be at least partially dependent upon the presence of potassium (3 mm in the incubation media). Reduction of the cytochromes attributable to potassium was inhibited by ouabain, indicating that intracellular potassium was the important variable. Addition of glucose or lactate to 'starved’ tissues was found to result in a complex cycle of oxidation and reduction of tissue NAD(P)H. A small initial reduction of NAD(P) was followed by an oxidation of NAD(P)H which occurred in an all-or-none fashion with reduction of the cytochromes. The oxidation of NAD(P)H and reduction of cytochrome b appeared to occur with a similar time course. Respiratory changes following addition of glucose were complex in time course, but established a new steady-state rate 0.41 μmol/g per min above the preaddition rate in 10–12 min. Despite a similar level of reduction in the cytochrome chain, stimulation of respiration by pyruvate was only about 50% of the rate observed with addition of glucose. However, stimulation of respiration by addition of equim concentrations of pyruvate and lactate was found to be additive, producing a 0.48 μmol/g per min increase in the steady-state rate of oxygen consumption. These data seem to support the conclusion that the cytoplasmic reducing equivalent derived from the initial oxidation of glucose or lactate plays an important, perhaps regulatory, role in the respiration of cerebral tissues.     

Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response

Glucose-stimulated insulin secretion is a multistep process dependent on beta-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters beta-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenase-dependent metabolism or a buffering of the NADH response by other metabolic mechanisms.     

SHIFTS IN UBIQUINONE REDOX STATUS OF RAT BRAIN IN VITRO WITH CATIONIC STIMULATION

–Changes in respiratory rate, ubiquinone (Q) redox status, lactate and pyruvate levels in chopped rat telencephalon were studied after cationic stimulation of respiration. When chopped telencephalon was incubated with glucose as substrate, increasing the K⁺ concentration from 5 mm to 55 mm was associated with a 57% increase in the respiratory rate and a reduction in Q redox status from 76% oxidized to 64% oxidized. Lactate increased by over 200%. Substitution of pyruvate for glucose with 5 mm -K⁺ resulted in a respiratory rate that was 78% of that seen with glucose and 55 mm -K⁺. Increasing K ⁺ from 5 mm to 55 mm increased the respiratory rate by only 6%, with pyruvate as substrate. Q underwent a reduction that was half that seen with glucose. Ouabain largely prevented the K ⁺-induced increase in respiratory rate with glucose as substrate, but Q was still reduced by 5 percentage points while lactate and pyruvate were unchanged. When respiration was uncoupled with 2,4-dinitrophenol, increasing the K⁺ concentration from 5 mM to 55 mm had no further effect on any of the metabolic parameters measured. Deletion of Ca²⁺ from the medium resulted in an increase in respiration of 18%, but neither the Q redox status nor the levels of lactate or pyruvate were significantly changed. The results demonstrate that the K⁺-induced stimulation of respiration results from a coordinated metabolic response whereas the stimulation of respiration associated with Ca²⁺ depletion is probably mediated through ion fluxes at the cell membrane and activation of Na⁺-K⁺-activated ATPases.     

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: a 31P-NMR and NAD(P)H fluorescence study

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO2), and extra-mitochondrial phosphates. Alterations in NADH and QO2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO2; (2) when QO2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO2. To investigate this further, effects of changing substrates at constant QO2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.     

DEVELOPMENTAL CHANGES IN GLYCOLYSIS IN RAT CEREBRAL CORTEX

Abstract— The ATP concentration in infant rat cerebral cortex slices which were incubated aerobically with glucose (5 m m ) as substrate was much higher than in those from the adult. The higher ATP concentration in slices from young rat was also obtained when they were incubated aerobically with pyruvate (10 m m ), dl -lactate (20 m m ) and dl -3-nydroxybutyrate (20 m m ) However, when the slices were incubated anaerobically with glucose, the ATP concentration was very low. Thus, the formation of ATP in the slices from the young rat was thought to be mainly due to their oxidative metabolism, as in those from the adult. The amounts of glycolytic key enzymes in rat cerebral cortex (hexokinase. phosphofructokinase and pyruvate kinase) increased with age. Glycolysis was actually shown to be less active in the cerebral slices from young rats than from the adult. In addition it is known that the tricarboxylic acid cycle enzymes in rat cerebrum also increase with age. Consequently, the activity with respect to ATP formation must be lower in the cerebral cortex slices from young rats than from the adult. The fact that ATP was nevertheless higher in the slices from young rats may be explained by a lower rate of degradation. Developmental increases in the amounts of Na⁺-K⁺-ATPase and Mg²⁺ -ATPase in rat cerebral cortex were greater than those of the glycolytic key enzymes. These are discussed in relation to the observation that the rate of aerobic glycolysis in slices from cerebral cortex of young rats was not increased by d -glutamate (5 m m ) and high potassium (50 m m ).     

Pancreatic islet beta-cells transiently metabolize pyruvate

Pancreatic beta-cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging. The observed redox changes, spatially separated between the cytoplasm and mitochondria, were compared with whole islet insulin secretion. As expected, both NAD(P)H and insulin secretion showed sustained increases in response to glucose stimulation. In contrast, pyruvate caused a much lower NAD(P)H response and did not generate insulin secretion. Low pyruvate concentrations decreased cytoplasmic NAD(P)H without affecting mitochondrial NAD(P)H, whereas higher concentrations increased cytoplasmic and mitochondrial levels. However, the pyruvate-stimulated mitochondrial increase was transient and equilibrated to near-base-line levels. Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. These data showed that glucose-stimulated mitochondrial NAD(P)H and insulin secretion are independent of pyruvate transport but dependent on NAD(P)H shuttling. In contrast, the pyruvate-stimulated cytoplasmic NAD(P)H response was enhanced by both inhibitors. Surprisingly the malate-aspartate shuttle inhibitor enabled pyruvate-stimulated insulin secretion. These data support a model in which glycolysis plays a dominant role in glucose-stimulated insulin secretion. Based on these data, we propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport.     

NADH-linked substrate dependence of peroxide-induced respiratory inhibition and calcium efflux in isolated renal mitochondria

Peroxide-induced state 3 respiratory inhibition and Ca2+ efflux in isolated renal mitochondria exhibited a NADH-linked substrate dependence. ADP-stimulated respiratory rates in the presence of various concentrations of tert-butyl hydroperoxide (tBOOH, 0-1000 nmol/mg protein) were determined using glutamate, beta-hydroxybutyrate, or pyruvate as substrates. Pyruvate-driven respiration was most sensitive to inhibition (Ki approximately equal to 75 nmol of tBOOH/mg protein) followed by beta-hydroxybutyrate and glutamate (Ki approximately equal to 150 nmol of tBOOH/mg protein for each). Calcium (5-10 nmol/mg protein) potentiated tBOOH-induced respiratory inhibition using all three substrates. Mitochondrial Ca2+ efflux, induced by tBOOH, was most pronounced with pyruvate as substrate. Glutamate prevented Ca2+ efflux while the efflux rate with beta-hydroxybutyrate was intermediate between glutamate and pyruvate. The substrate-dependent pattern of tBOOH-induced NAD(P)H (NADH plus NADPH) and cytochrome b oxidation was similar to that seen for respiratory inhibition and Ca2+ efflux suggesting that NAD(P)H may be a common factor in both responses. Low tBOOH concentrations inhibited pyruvate dehydrogenase flux while higher concentrations enhanced pyruvate dehydrogenase flux and activation. The results are discussed in relation to currently proposed theories of reactive oxygen-induced respiratory inhibition, Ca2+ efflux, and reperfusion injury.     

Integrating cytosolic calcium signals into mitochondrial metabolic responses.

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glucose-glycerol mixtures

Summary The metabolism of C. butyricum was manipulated, at neutral pH and in carbon limited chemostat cultures by changing the overall degree of reduction of the substrate, using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate and lactate). When the glycerol (in C moles)/glucose+glycerol (in C moles) ratio was progressively changed from 0 to 1 a corresponding increase of 1,3-propanediol production occured and an immediate and drastic decrease of the specific rate of acetate production was observed while the specific rate of butyrate production only decreased slightly. For glycerol (in C moles)/glucose+glycerol (in C moles) ratios higher than 0.5, the q_{NAD(P)H from Fd} and the CO₂/H₂ molar ratio increased sharply, the first becoming positive and the second higher than 1. This indicates a complete reversion of the electron flow: part of reduced ferredoxin produced by the phosphoroclastic cleavage of pyruvate to acetyl-CoA was diverted from H₂ formation toward NAD(P) reduction by the ferredoxin-NAD(P) reductase(s) in order to produce NAD(P)H. This change in the electron flow was associated to an increase in the specific rate and the yield of 1,3-propanediol production related to glycerol.     

Diversion of the metabolic flux from pyruvate dehydrogenase to pyruvate oxidase decreases oxidative stress during glucose metabolism in nongrowing Escherichia coli cells incubated under aerobic, phosphate starvation conditions

Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (P(i)). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in DeltaahpCF mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, P(i) starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H(2)O(2) by NADH dehydrogenase in the respiratory chain. The residual low levels of H(2)O(2) produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H(2)O(2) may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.     

Anoxia-Induced Changes in Pyridine Nucleotide Redox State in Cortical Neurons and Astrocytes

NAD(P)H autofluorescence was used to verify establishment of metabolic anoxia using primary cultures of cortical neurons and astrocytes. Cells on cover slips were placed in a chamber and O₂ was displaced by continuous infusion of argon. Perfusion with medium at PO₂ < 0.4 mm Hg caused an increase in NAD(P)H fluorescence, albeit to levels lower than that obtained with cyanide. Addition of the nitric oxide-generating agent DETA-NO to the hypoxic medium further increased fluorescence to the level with cyanide. Fluorescence under anoxia remained high in the presence of glucose, but declined in neurons and not in astrocytes when glucose was substituted with 2-deoxyglucose. Reoxygenation of neurons resulted in a decline in fluorescence and a loss in fluorescent gradient between fully reduced and fully oxidized (plus respiratory uncoupler). We conclude that (1) DETA-NO is useful for generating metabolic anoxia in the presence of argon (2) Exogenous glucose is necessary to maintain NAD(P)H in a reduced state during metabolic anoxia in neurons but not astrocytes (3) Neurons undergo a partially irreversible decline in NAD(P)H fluorescence during metabolic anoxia and reoxygenation that could contribute to prolonged metabolic failure. Special issue dedicated to John P. Blass.     

RESPIRATION IN VITRO OF SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

Abstract— —(1) The respiratory properties of synaptosomes and mitochondria from mammalian cerebral cortex are compared.
(2) Synaptosome showed high and linear respiration with glucose and pyruvate as substrates in Krebs-Ringer media. Mitochondria showed such respiration only with pyruvate as substrate in media lacking Na and high in K and phosphate.
(3) Exposure of synaptosomes to hypotonic media caused loss of lactate dehydrogenase (LDH) and protein, and respiration diminished and became non-linear.
(4) Both ATP and phosphocreatine were synthesised by synaptosomes with glucose as substrate. ATP was synthesised by mitochondria in the presence of pyruvate.
(5) Synaptosome but not mitochondria showed some capacity for active accumulation of potassium.
(6) Both mitochondria and synaptosomes respired with glutamate as substrate. Glutamate caused 80 per cent loss of ATP and phosphocreatine in synaptosomes but did not diminish the level of mitochondrial ATP.     

EXCHANGE OF TAURINE IN BRAIN SLICES OF ADULT and 7-DAY-OLD RATS

Abstract— The influx or efflux of taurine in brain slices prepared from adult and 7-day-old rats was studied in Krebs-Ringer bicarbonate-glucose medium with 0,2 and 10 m m -taurine. The exchange of taurine between the slices and the medium was slow, and no steady-state concentration was reached within the experimental period of 150 min. In both experimental groups there was a net influx of taurine into the slices from 10 m m -taurine and a slight net efflux from the slices into 2 m m -taurine. The rate of influx from 10 m m -taurine was about the same in the two groups after an initial period of faster influx into the slices of adult rats. There was some rapid initial efflux into 0 and 2 m m -taurine solutions from the slices from 7-day-old rats, but with prolonged incubation these slices were better able to maintain their intracellular taurine than the slices from adult rats. The reasons and significance of the high cerebral concentration of taurine in immature brain in vivo are briefly discussed in the light of the present and earlier studies.     

Plasma membrane potential oscillations in insulin secreting Ins-1 832/13 cells do not require glycolysis and are not initiated by fluctuations in mitochondrial bioenergetics

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Δψ(p) oscillatory mechanism is initiated.     

Respiratory control in the glucose perfused heart. A 31P NMR and NADH fluorescence study

The phosphate metabolites, adenosine diphosphate (ADP), inorganic phosphate (Pi), and adenosine triphosphate (ATP), are potentially important regulators of mitochondrial respiration in vivo. However, previous studies on the heart in vivo and in vitro have not consistently demonstrated an appropriate correlation between the concentration of these phosphate metabolites and moderate changes in work and respiration. Recently, mitochondrial NAD(P)H levels have been proposed as a potential regulator of cardiac respiration during alterations in work output. In order to understand better the mechanism of respiratory control under these conditions, we investigated the relationship between the phosphate metabolites, the NAD(P)H levels, and oxygen consumption (Q02) in the isovolumic perfused rat heart during alterations in work output with pacing. ATP, creatine phosphate (CrP), Pi and intracellular pH were measured using 31P NMR. Mitochondrial NAD(P)H levels were monitored using spectrofluorometric techniques. Utilizing glucose as the sole substrate, an increase in paced heart rate led to an increase in Q02 from 1.73 +/- 0.09 to 2.29 +/- 0.12 mmol Q2/h per g dry wt. No significant changes in the levels of Pi, PCr, ATP, or the calculated ADP levels were detected. Under identical conditions, an increase in heart rate was associated with a 23 + 3% increase in NAD(P)H fluorescence. Thus, under the conditions of these studies, an increase in Q02 was not associated with an increase in ADP or Pi. In contrast, increases in Q02 were associated with an increase in NAD(P)H. These data are consistent with the notion that increases in the mitochondrial NADH redox state regulate steady-state levels of respiration when myocardial work is increased.     

Linear growth and poly(beta-hydroxybutyrate) synthesis in response to pulse-wise addition of the growth-limiting substrate to steady-state heterotrophic continuous cultures of Aquaspirillum autotrophicum.

Heterotrophic pyruvate-limited steady-state continuous cultures of the bacterium Aquaspirillum autotrophicum were perturbed with a pulse injection of a small volume of concentrated pyruvate solution. These cultures exhibited an instantaneous change in the growth dynamics, turning from steady state to apparently linear growth. These transient growth-responses had no lag phase and were clearly distinct from unlimited exponential growth according to the initial rates of increase of biomass and substrate disappearance kinetics. A linear accumulation with time of poly(beta-hydroxybutyrate) was observed within the cells. Slopes of these linear responses were negatively correlated with the dilution rate. Physiological bases of linear growth are discussed in the light of the models of H. E. Kubitschek. Poly(beta-hydroxybutyrate) synthesis in the absence of exogenous limitation may serve to protect the cells against a transient metabolic overflow.     

Effects of homocysteine on metabolic pathways in cultured astrocytes

Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes.     

NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes

A fiber-optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic-anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose-depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine-depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate-level phosphorylation, as shown by decreases of 35-52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6-38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.