EARLY CHANGES IN RESPIRATION, AEROBIC GLYCOLYSIS AND CELLULAR NAD(P)H IN SLICES OF RAT CEREBRAL CORTEX EXPOSED TO ELEVATED CONCENTRATIONS OF POTASSIUM

Abstract— The initial effects of an elevated potassium concentration (30 m m ) on the energy metabolism of incubated slices of rat cerebral cortex have been examined using spectrophotometric and polarographic techniques. Respiratory responses to additions of potassium were found to be definitely limited in time. This response was followed by an increase in the rate of aerobic glycolysis. Slice NAD(P)H and cytochrome b paralleled this metabolic sequence by exhibiting an initial oxidation followed by a net increase in the steady-state levels of reduced intermediates, particularly in the case of NAD(P)H. Substitution of pyruvate (10 m m ) for glucose in the standard incubation media produced significant alterations in the respiratory responses to the addition of potassium. Although the period of increased oxygen consumption was again limited it was somewhat greater in magnitude and significantly prolonged in time relative to changes observed with glucose as substrate. Changes in steady-state levels of NAD(P)H were altered similarly and the net increase of NAD(P)H was not observed with pyruvate as substrate. We suggest that the metabolic responses of brain slices to increased potassium do not involve simultaneous activation of the respiratory and glycolytic pathways as has been previously assumed. Rather, a distinctly biphasic response is observed reminiscent of the Crabtree effect observed in other systems.     

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.     

Respiratory control in isolated perfused rat heart. Role of the equilibrium relations between the mitochondrial electron carriers and the adenylate system.

The effects of KCl-induced cardiac arrest on the redox state of the fluorescent flavoproteins and nicotinamide nucleotides and on that of cytochromes c and a were studied by surface fluorometric and reflectance spectrophotometric methods. These changes were compared with measurements of the concentrations of the adenylate system, creatine phosphate, some intermediates of the tricarboxylic acid cycle and reactants of the glutamate dehydrogenase system. KCl-induced cardiac arrest caused reduction of the fluorescent flavoproteins and nicotinamide nucleotides, oxidation of cytochromes c and a, inhibition of oxygen consumption and an increase in the ATP/(ADP X Pi) ratio. The increase in the latter was due mainly to a decrease in the concentration of Pi and an equivalent increase in creatine phosphate. The cytochromes c and a were maintained at equal redox potential and changed in parallel. When the redox state of the mitochondrial NAD couple was calculated from the glutamate dehydrogenase equilibrium, the free energy change (deltaG) corresponding to the potential difference between the NAD couple and cytochrome c was 115.8 kj/mol in the beating heart and 122.2 kj/mol in the arrested heart. The deltaG values of ATP hydrolysis calculated from the concentrations of ATP, Pi and ADP, corrected for bound ADP, were 111.1 kj/2 mol and 115.4 kj/2 mol in the beating and arrested heart respectively. The accumulation of citrate and the direction of the redox changes in the respiratory carriers indicate that the tricarboxylic acid cycle flux is controlled by the respiratory chain. The data also show a near equilibrium between the electron carriers and the adenylate system and suggest that the equilibrium hypothesis of mitochondrial respiratory control is applicable to intact myocardial tissue.     

Respiratory control in isolated perfused rat heart. Role of the equilibrium relations between the mitochondrial electron carriers and the adenylate system

The effects of KCl-induced cardiac arrest on the redox state of the fluorescent flavoproteins and nicotinamide nucleotides and on that of cytochromes c and a were studied by surface fluorometric and reflectance spectrophotometric methods. These changes were compared with measurements of the concentrations of the adenylate system, creatine phosphate, some intermediates of the tricarboxylic acid cycle and reactants of the glutamate dehydrogenase system.

KCl-induced cardiac arrest caused reduction of the fluorescent flavoproteins and nicotinamide nucleotides, oxidation of cytochromes c and a, inhibition of oxygen consumption and an increase in the ATP/(ADP × P_i) ratio. The increase in the latter was due mainly to a decrease in the concentration of P_i and an equivalent increase in creatine phosphate. The cytochromes c and a were maintained at equal redox potential and changed in parallel. When the redox state of the mitochondrial NAD couple was calculated from the glutamate dehydrogenase equilibrium, the free energy change (ΔG) corresponding to the potential difference between the NAD couple and cytochrome c was 115.8 kJ/mol in the beating heart and 122.2 kJ/mol in the arrested heart. The ΔG values of ATP hydrolysis calculated from the concentrations of ATP, P_i and ADP, corrected for bound ADP, were 111.1 kJ/2 mol and 115.4 kJ/2 mol in the beating and arrested heart respectively.

The accumulation of citrate and the direction of the redox changes in the respiratory carriers indicate that the tricarboxylic acid cycle flux is controlled by the respiratory chain. The data also show a near equilibrium between the electron carriers and the adenylate system and suggest that the equilibrium hypothesis of mitochondrial respiratory control is applicable to intact myocardial tissue.     

Kinetic advantage of the interaction between the fatty acid beta-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C4, C8 and C14)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD+ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 microM in gently sonicated and 15 microM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD(+)-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD+ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of beta-oxidation enzymes in situ. Respiration-linked oxidation of butyryl-, octanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

CONTROL OF AEROBIC GLYCOLYSIS AND PYRUVATE KINASE ACTIVITY IN CEREBRAL CORTEX SLICES

Abstract— —The concentrations of glycolytic intermediates were measured in aerobically incubated guinea pig cerebral cortex slices. Increasing the concentration of potassium in the medium increased fructose diphosphate ten-fold and triose phosphates three-fold. Omitting calcium increased all the glycolytic intermediates except pyruvate; triose phosphates were increased most. The changed patterns of the glycolytic intermediate profile in the slices are consistent with the previously proposed hypothesis that the phosphofructokinase is a main regulatory step in glycolysis. Glycolysis is also limited at the step of pyruvate kinase, which is inhibited in cerebral cortex slices. Calcium in the tissue and cellular organization of the slices were shown to be responsible for this inhibition. It was concluded that the effects of potassium and calcium on aerobic glycolysis in cerebral cortex slices are direct—on the pyruvate kinase—and also indirect. Calcium was shown to be inhibitive also to the activities of hexokinase, phosphoglucoisomerase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase and enolase of guinea pig cerebral tissue.     

Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.     

Effects of HCO3/CO2 on the cytochrome redox potential and metabolic responses of isolated rat cerebral cortex.

The importance of HCO₃^?/CO₂ to the maintenance of stable metabolic conditions in rat cerebral cortex slices has been investigated. Replacement of bicarbonate buffered media with glycylglycine or phosphate buffered salines resulted in an increased lability of the cytochrome redox potential of brain slices as measured by dual wavelength spectroscopy. Depletion of reduced cytochrome in the electron transport chain was associated with changes in the metabolic responses of tissues to electrical stimulation or elevated concentrations of potassium. This appears primarily as a loss of the late reductive responses of the tissue nicotinamide adenine dinucleotides NAD(P)H to these stimuli and decreased lactic acid output of the tissues. The effects could be largely reversed in a combined glycylglycine and HCO₃^?/CO₂ buffered media. It is suggested that although the reduction of NAD(P) in response to stimulation may be substantially located in the cytosol, it is also modulated by the mitochondrial redox potential. It is suggested that the lability of the redox potential in the absence of HCO₃^?/CO₂ may be related to depletion of TCA cycle intermediates normally replaced by CO₂ fixation.     

STUDIES ON THE REGULATION OF GABA SYNTHESIS: THE INTERACTION OF ADENINE NUCLEOTIDES AND GLUTAMATE WITH BRAIN GLUTAMATE DECARBOXYLASE

The effects of adenine nucleotides and glutamate on glutamate decarboxylase were studied in a dialyzed, high-speed supernatant of rat brain. When incubated with 10 μm -pyridoxal-P the enzyme was strongly inhibited by ATP, ADP and their Mg²⁺ complexes at concentrations which were well below tissue levels. The enzyme was not significantly inhibited by 15 mm -AMP or by 100 μM-3′-5’cyclic AMP or 3′-5’cyclic GMP. Inhibition by the nucleotides cannot be described in conventional steady-state kinetic terms. Addition of ATP in the presence of pyridoxal-P resulted in a slow, progressive decrease in the reaction rate which was similar to the inactivation observed when the enzyme was incubated in the absence of pyridoxal-P. The progressive inactivation in the presence of ATP was minimal at concentrations of glutamate which were well below K_m and became much more pronounced at higher glutamate concentrations. Addition of suprasaturating amounts of pyridoxal-P late in the incubation when the enzyme was almost completely inactivated resulted in an immediate and complete reactivation of the enzyme. Inhibition by ATP could be prevented by addition of saturating amounts of pyridoxal-P at the start of the reaction and was also relieved by addition of potassium phosphate buffer. The results suggest that inhibition by the nucleotides involves the prior formation of the inactive apoenzyme which results from the glutamate-promoted dissociation of pyridoxal-P. In the absence of the nucleotides, the enzyme is normally reactivated by the added pyridoxal-P. The nucleotides act to block this reassociation of pyridoxal-P with the apoenzyme thereby producing a progressive inactivation of the enzyme. The implications of these results for the regulation of GABA synthesis are discussed.     

THE UPTAKE OF AMINO ACIDS BY THE INTACT OLFACTORY BULB OF THE MOUSE: A COMPARISON WITH TISSUE SLICE PREPARATIONS

Abstract— Intact olfactory bulbs from 8- to 15-day-old mice were compared to slices of olfactory bulb and cerebral hemisphere with respect to uptake of amino acids, respiratory rate, levels of ATP, retention of sodium and potassium, and extracellular space. The uptake of amino acids was lower in intact bulbs than in slice preparations, both in regard to initial rates of uptake and to final steady state levels, at external amino acid concentrations from 0·2 to 2·0mM. Uptake was lower in bulbs attached to brain than in those separated from it and somewhat higher in the half of the bulb closer to the cut surface. In all preparations the uptake of glutamic acid and glycine was highest, uptake of histidine and valine was intermediate, and uptake of lysine was lowest. These differences between intact bulbs and slices could not be correlated with differences in respiratory rate, levels of ATP, or changes in levels of Na⁺ or K⁺ ions. Increases in dextran and inulin spaces, however, were greatest in preparations having the highest rates of amino acid uptake. Although for several amino acids the maximal velocity of uptake (V_max) was 4-fold higher in slices of bulb than in intact bulbs, the affinity of amino acids to their carrier systems ( K _m) was similar, an indication that the same transport process was operative in both cases. On the basis of these results we propose that intact olfactory bulbs incubated in vitro possess a regulatory mechanism for the limitation of amino acid uptake that is absent or diminished in slices.     

IONS AND THE TRANSPORT OF GAMMA-AMINOBUTYRIC ACID BY SYNAPTOSOMES

Abstract— The initial rate of uptake of [2,3-³H]gamma-aminobutyric acid by rat brain synaptosomes was studied under incubation conditions in which GABA metabolism was minimal. The presence of both sodium and potassium in the incubation medium was essential for sustained uptake. Uptake proceeded for a short period of time in the absence of potassium and then ceased. No uptake was observed when sodium chloride was completely replaced with sucrose or choline chloride. The sodium-dependence curve for GABA uptake was markedly sigmoid. The sigmoid character of the curve was not attributable to a lag phase in uptake at low sodium concentrations. Calcium strongly stimulated the initial rate of uptake at low sodium concentrations but had little effect at sodium concentrations above 100 m m and was not able to support uptake in the absence of sodium. The sigmoid character of thesodium-dependence curve was completely eliminated by 20 m m calcium ion. Magnesium and phosphate had little effect on the initial rate of GABA uptake.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Use of noninvasive fluorometry and spectrophotometry to study epithelial metabolism and transport

Various examples illustrating the use of spectrophotometry and fluorometry in epithelia are presented. The first example uses the redox level of cytochrome aa3, measured spectrophotometrically as an index of tissue anoxia in cortical tubules and slices from the rabbit kidney. In the second example the redox level is used to measure the kinetics of aerobic energy production during transition to anoxia in the midgut of the tobacco hornworm. In the third application, the redox level of mitochondrial NADH is measured fluorometrically in a cortical tubule suspension from the rabbit kidney. Inhibition of active transport work causes reduction of NAD whereas increased work elicits oxidation of NAD, both occurring as expected from mitochondrial transitions to a lesser or more active state, respectively. Another use of NADH fluorescence is the determination of the relative effectiveness of metabolic substrates to deliver reducing equivalents to the respiratory chain in a particular tissue. Redox changes in mitochondrial NAD may be used to distinguish between primary metabolic and primary transport effects of hormones, drugs, and changes in the state of the organism. Finally, examples are provided of the use of an intracellular pH-sensitive dye and an extracellular calcium-sensitive dye in kidney tubules.     

The Kinetics of Pyridine Nucleotide Reduction following the Addition of Glucose to Bakers' Yeast

Changes of pyridine nucleotide concentration in yeast followingthe addition of glucose have been measured by a fluorimetricassay applied to cell extracts. The major changes were in nicotinamideadenine dinucleotide (NAD) which, during the first minute afterglucose addition, was rapidly reduced and then more slowly reoxidized.Later, one to two minutes after adding glucose, NAD was reducedagain to a steady-state value. No NAD was reduced when acetaldehydewas added simultaneously with glucose. The oxidation of NAD,which followed the initial reduction, was slowed down by sodiumfluoride and almost completely abolished by sodium sulphite.The initial reduction of NAD is ascribed to 3-phosphoglyceraldehydedehydrogenase and its subsequent oxidation is ascribed to alcoholdehydrogenase. Probably only that NAD bound to 3-phosphoglyceraldehydedehydrogenase is reduced initially. The reasons for these viewsare discussed.     

Evaluation of electron-transfer flavoprotein and alpha-lipoamide dehydrogenase redox states by two-channel fluorimetry and its application to the investigation of beta-oxidation

A new method permitting the simultaneous evaluation of the redox states of alpha-lipoamide dehydrogenase and electron-transfer flavoprotein in intact rat liver mitochondria by two-channel fluorimetry is described. It is shown that correction for the partial overlap of emission spectra can readily be introduced after a calibration procedure is performed. This method was applied to the investigation into regulation of palmitoylcarnitine oxidation. It was found that in the presence of rotenone, malonate and a redox buffer for the mitochondrial NAD-system, the beta-oxidation flux was sensitive to variations in redox state of respiratory chain electron carriers at low states of NAD reduction. Therefore, the concept of beta-oxidation control caused solely by the NAD redox state can no longer be sustained.     

Effects of guanidine derivatives on mitochondrial function

Steady-state concentrations of citric acid cycle compounds accumulating during state 3 oxidation of pyruvate by guinea pig heart mitochondria have been measured using isotopic and fluorometric techniques; incubations partially inhibited with several guanidine derivatives and other inhibitors were compared with controls. The changes in levels of intermediates which occurred with guanidine derivatives were quantitatively and qualitatively identical to those with amytal; this pattern of intermediates was therefore not limited to those compounds which possess hypoglycemic propertiesin vitro. With antimycin, rotenone and nigericin the pattern of intermediates was specific for each agent, and differed from that with guanidine derivatives and amytal.Reduced pyridine nucleotides were also estimated at progressively increasing degrees of respiratory inhibition by these same agents. Lower concentrations of phenethylbiguanide and amytal produced identical increases in state 3 level of reduced pyridine nucleotide, while higher phenethylbiguanide concentrations were associated with a phosphatedependent decrease in reduced pyridine nucleotide level in both state 3 and state 4 which was not observed with amytal, and not accompanied by a stimulation of respiratory rate. These changes resemble the metabolic condition termed state 6, and are consistent with a calciumlike activity of guanidine derivatives. Changes in level of reduced pyridine nucleotide were observed which were specific to rotenone and nigericin; these changes, combined with the patterns of citric acid cycle intermediates observed with these inhibitors, are useful in interpreting the effects of these agents on the functional state of intact mitochondria.     

The responses of rat hepatocytes to glucagon and adrenaline. Application of quantified elasticity analysis.

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6-phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6-phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.     

Effects of calcium on mitochondrial NAD(P)H in paced rat ventricular myocytes.

The response of the steady-state level of mitochondrial NAD(P)H of individual cardiac myocytes to substrate and to pharmacological alteration of intracellular calcium was investigated using a defined pacing protocol. Rapid pacing (5 Hz) reversibly decreased the NAD(P)H level and increased oxygen consumption whereas phosphocreatine and ATP levels did not change significantly. Verapamil plus NiCl2 blockade of calcium channels abolished contractions. Ryanodine, which prevents calcium-induced calcium release, also stopped cell contraction. NAD(P)H levels do not change in the absence of contraction. Blockade of sarcolemmal K+ channels did not stop contraction, and NAD(P)H levels reversibly decreased during rapid pacing. Thus rapid contractions are associated with a reversible decrease in NAD(P)H levels. Ruthenium red blockade of Ca2+ entry into mitochondria did not block contraction but significantly decreased NAD(P)H levels in both slowly paced (0.5 Hz) and rapidly paced cells. The simplest explanation of these data is that the steady-state reduction of NAD(P)H is strongly dependent on the rate of ATP utilization and not on sarcoplasmic Ca2+ levels when the oxygen and substrate supplies are not limiting and the intracellular calcium regulation is maintained. An effect of intracellular Ca2+ on NAD(P)H is observed only when Ca2+ entry into mitochondria is blocked with ruthenium red.     

UPTAKE OF CALCIUM BY SUBCELLULAR FRACTIONS ISOLATED FROM OUABAIN-TREATED CEREBRAL TISSUES

Abstract— Slices of cerebral cortex were incubated in medium containing 0·75 or 2·8 mM ⁴⁵CaCl₂, in the presence or absence of 0·01–0·1 m m -ouabain. Ouabain induced accumulation of calcium by slices to a maximum of 4 μmoles/g of tissue/hr (0·75 m m -CaCl₂ in the medium) and to 8 μmoles/g of tissue/hr (2·8 m m -CaCl₂ in the medium). Accumulation of Ca²⁺ occurred more slowly than loss of K⁺ from the slices and more closely resembled the pattern of Na⁺ uptake.
Mitochondrial fractions isolated from ouabain-treated slices contained significantly more calcium than controls. Inclusion of EDTA in the homogenization medium resulted in decreased amounts of particulate-bound calcium.
The effect of ouabain on accumulation of calcium is discussed with regard to possible relationships to processes of active and passive transport.     

NAD: Not just a pawn on the board of plant-pathogen interactions

Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections.