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1.
Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i., [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+], and [Na+]., The increase in [Na+], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.  相似文献   

2.
The present experiments were carried out to investigate the effect of increased fluxes of H+ across the plasmalemma on glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum L. (1) Increased H+ influx was produced by adding glucose, 6-deoxyglucose, 2-deoxyglucose, or sodium fluoride. The net influx decreased to zero after 3 min. This recovery was accompanied by an increase in the rate of O2 uptake, but not of dark CO2 fixation. When glucose or fluoride were added, the increase of O2 uptake occurred without a decrease in the ATP/ADP ratio, and was large enough to provide the ATP that would be needed for compensatory H+ extrusion via the plasmalemma H+-ATPase. When 2-deoxyglucose was added, the rise of respiration was restricted by sequestration of phosphate and depletion of phosphorylated metabolites, the ATP/ADP ratio declined, and a slow net H+ influx started again after 4 min. (2) Alkalinisation of the medium to induce an H+ efflux resulted in rapid activation of dark CO2 fixation, but not of O2-uptake. (3) A stimulation of respiration or dark CO2 fixation was always accompanied by a decrease of phosphoenolpyruvate. This shows that the primary sites for regulation of glycolysis are pyruvate kinase and phosphoenolpyruvate carboxylase, respectively. (4) There was no consistent relation between glycolytic flux and triose-phosphates or hexose-phosphates. This shows that the reactions involved in carbohydrate mobilisation and the conversion of hexose-phosphates to triose-phosphates only have a secondary role in stimulation of glycolysis. (5) Phosphofructokinase will be stimulated as a consequence of the decrease in phosphoenolpyruvate. (6) The increase in glycolytic flux occurred independently of (in the case of 2-deoxyglucose and fluoride), or before (in the case of glucose), any increase of fructose-2,6-bisphosphate. When fructose-2,6-bisphosphate did increase (after supplying glucose), this was accompanied by an increase of triose-phosphate and fructose-1,6-bisphosphate, which otherwise remained very low. It is argued that fructose-2,6-bisphosphate increases as a consequence of the decrease of glycerate-3-phosphate, a known inhibitor of the synthesis of this regulator metabolite. However, activation of pyrophosphate fructose-6-phosphate phosphotransferase by fructose-2,6-bisphosphate does not play an obligatory role in the stimulation of glycolysis.  相似文献   

3.
We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H+-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F0F1-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F0F1-ATPase and plasma membrane H+-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.  相似文献   

4.
5.
Respiration and glycolysis in the human diploid cell strain WI-38   总被引:6,自引:0,他引:6  
Assessment of the respiratory and glycolytic capacity of non-growing WI-38 cells shows that, in the absence and presence of added glucose, the mean rates of oxygen consumption were 247 (QO2 = 5.61) and 208 (QO2 = 4.73) mμmoles/mg dry wt/hr., respectively. Mean glucose consumption was 225 mμmoles/mg dry wt/ hr. With uniformly labeled 14C glucose as substrate, 36 mμg atoms of carbon dioxide were produced, corresponding to 15–20% of the total cellular respiration. Mean values for lactate production in the presence and absence of glucose were 345 (QLO2 = 7.85) and 196 (QLO2 = 4.45) mμmoles/mg dry wt/hr., respectively. Human diploid cells in culture age, in the sense that their ability to proliferate decreases with time during serial subcultivation. Studies of their respiratory and glycolytic capacity as a function of the aging process showed that total respiration, glucose respiration and glycolytic capacity were relatively constant for cells in the middle and late passages and indicate that aging in this sense is not directly related to the respiratory and glycolytic capacity of the cell.  相似文献   

6.
Oxygen consumption for bioenergetic purposes has long been thought to be the prerogative of mitochondria. Nevertheless, mitochondrial gene knockout (ρ0) cells that are defective in mitochondrial respiration require oxygen for growth and consume oxygen at the cell surface via trans-plasma membrane electron transport (tPMET). This raises the possibility that cell surface oxygen consumption may support glycolytic energy metabolism by reoxidising cytosolic NADH to facilitate continued glycolysis. In this paper we determined the extent of cell surface oxygen consumption in a panel of 19 cancer cell lines. Non-mitochondrial (myxothiazol-resistant) oxygen consumption was demonstrated to consist of at least two components, cell surface oxygen consumption (inhibited by extracellular NADH) and basal oxygen consumption (insensitive to both myxothiazol and NADH). The extent of cell surface oxygen consumption varied considerably between parental cell lines from 1% to 80% of total oxygen consumption rates. In addition, cell surface oxygen consumption was found to be associated with low levels of superoxide production and to contribute significantly (up to 25%) to extracellular acidification in HL60ρ0 cells. In summary, cell surface oxygen consumption contributes significantly to total cellular oxygen consumption, not only in ρ0 cells but also in mitochondrially competent tumour cell lines with glycolytic metabolism.  相似文献   

7.
AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O2 deficiency glycolytic degradation of glucose leads to formation of lactate and H+, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H+ export. The present study explored whether AMPK influences Na+/H+ exchanger (NHE) activity and/or Na+-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pHi) was estimated utilizing BCECF fluorescence and Na+/H+ exchanger activity from the Na+-dependent re-alkalinization (ΔpHi) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pHi and ΔpHi in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pHi and ΔpHi was blunted in the presence of the Na+/H+ exchanger inhibitor cariporide (10 μM), but not by the H+ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.  相似文献   

8.

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.  相似文献   

9.
Isotope discrimination is a common feature of biosynthesis in nature, with the result that different classes of carbon compounds frequently display different 13C/12C ratios. The 13C/12C ratio of lipid in potato tuber tissue is considerably lower than that for starch or protein. We have collected respiratory CO2 from potato discs in successive periods through 24 hr from the time of cutting—an interval in which the respiration rate rises 3–5-fold. The 13C/12C ratio of the evolved CO2 was determined for each period, and compared with the 13C/12C ratios of the major tissue metabolites. In the first hours the carbon isotope ratio of the CO2 matches that of lipid. With time, the ratio approaches that typical of starch or protein. An estimation has been made of the contribution of lipid and carbohydrate to the total respiration at each juncture. In connection with additional observations, it was deduced that the basal, or initial, respiration represents lipid metabolism—possibly the α-oxidation of long chain fatty acids—while the developed repiration represents conventional tricarboxylic acid cycle oxidation of the products of carbohydrate glycolysis. The true isotopic composition of the respiratory CO2 may be obscured by fractionation attending the refixation of CO2 during respiration, and by CO2 arising from dissolved CO2 and bicarbonate preexisting in the tuber. Means are described for coping with both pitfalls.  相似文献   

10.
With glucose as a substrate, the oxygen consumption in yeast in inhibited by 2· 10-5M ethylene diisothiocyanate. The degree of inhibition was only to a small extent dependant on pH. Radiorespirometric experiments with uniformely labelled glucose showed that the CO2-production from glucose increased, probably due to increased glycolytic activity. Conversion of C-1 to CO2 was unaffected by the inhibitor, while the evolution of CO2 from C-6 was strongly inhibited. The same was the case with CO2 from C-1 in acetate. Respiration of ethanol was more strongly inhibited than that of glucose or acetate. Experiments with dual wavelength spectrophotometry showed the inhibition to be located on the Krebs cycle side of the respiratory flavoproteins. It is concluded that the action of ethylene diisothiocyanate on respiration must be located at the mitochondria.  相似文献   

11.
Soil acidification is a very important process in the functioning of earth's ecosystems. A major source of soil acidity is CO2, derived from the respiration of plant roots and microbes, which forms carbonic acid in soil waters. Because elevated atmospheric CO2 often stimulates respiration of soil biota in experiments that test ecosystem effects of elevated atmospheric CO2, we hypothesize that rising atmospheric CO2 (which has increased from ~200 ppm since the interglacial and may exceed 550 ppm by the end of the 21st century) is significantly increasing acid inputs to soils. Here, using column‐leaching experiments with contrasting soils, we demonstrate that soil CO2 is a much more potent agent of soil acidification than is generally appreciated, capable of displacing almost all exchangeable base cations in soils, and even elevating Al(III) concentrations in H2CO3‐acidified soil waters. The potent soil acidifying potential of soil H2CO3 is attributed to the low pKa,1 of molecular H2CO3 (3.76 at 25°C), which contrasts greatly with that of (a convention that combines CO2 (aq) and molecular H2CO3, the pKa,1 of which is 6.36 at 25°C). This distinction is significant for soil systems because of soil's greatly elevated CO2, their variety of sinks for H+, and the wide range of contact times between soil solids, water, and gas. Modelling suggests that a doubling of atmospheric CO2 may increase acid inputs from carbonic acid leaching by up to 50%. Combined with the results of CO2 studies in whole ecosystems, this implies that increases in atmospheric CO2 since the interglacial have gradually acidified soils, especially poorly buffered soils, throughout the world.  相似文献   

12.
Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H2O2) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H2O2 derives from superoxide (O2˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O2˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O2˙̄ and H2O2 may provide feedback signals to modulate mitochondrial metabolism.  相似文献   

13.
The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
15.
16.
High-throughput applicable screens for identifying drug-induced mitochondrial impairment are necessary in the pharmaceutical industry. Hence, we evaluated the XF96 Extracellular Flux Analyzer, a 96-well platform that measures changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells. The sensitivity of the platform was bench-marked with known modulators of oxidative phosphorylation and glycolysis. Sixteen therapeutic agents were screened in HepG2 cells for mitochondrial effects. Four of these compounds, thiazolidinediones, were also tested in primary feline cardiomyocytes for cell-type specific effects. We show that the XF96 platform is a robust, sensitive system for analyzing drug-induced mitochondrial impairment in whole cells. We identified changes in cellular respiration and acidification upon addition of therapeutic agents reported to have a mitochondrial effect. Furthermore, we show that respiration and acidification changes upon addition of the thiazoldinediones were cell-type specific, with the rank order of mitochondrial impairment in whole cells being in accord with the known adverse effects of these drugs.  相似文献   

17.
Heterotrophic activity in macroalgae has been little studied, but the red macroalga Grateloupia doryphora is known to grow in light at a higher rate in a glycerol-containing medium than in seawater. The effects of 0·1 M exogenous glycerol in seawater (SW90-gly) on the respiration rate of G. doryphora and the role played by light were investigated. The algae pretreated for 2 h in the light and in SW90-gly evolved oxygen and fixed carbon dioxide (H14CO3 ?), but also evolved radioactive 14CO2 from [14C]glycerol. The rate of oxygen evolution was lower than that of samples in seawater, due to a high respiration rate and/or a partial inhibition of photosynthesis induced by glycerol. In contrast, the rate of inorganic carbon fixation was higher in SW90-gly than in control samples in seawater, suggesting that non-photosynthetic patterns were operating. In darkness, after pretreatment in the light in SW90-gly, samples showed a high oxygen uptake rate just after the light was turned off. Twenty minutes of darkness were enough to decrease this high respiration rate to that of samples in seawater. The oxygen uptake observed in all experiments with glycerol was mitochondrial as it was inhibited by potassium cyanide and salicylhydroxamic acid (SHAM). Pretreatment of samples in the light in SW90-gly with the photosynthetic inhibitor DCMU did not inhibit ensuing dark respiration, thus providing evidence for a non-photosynthetic effect of the light. The highest dark respiration rate was observed after the samples were pretreated in monochromatic blue light in glycerol-containing media.  相似文献   

18.
Cancer cell metabolism is largely controlled by oncogenic signals and nutrient availability. Here, we highlighted that the glucocorticoid-induced leucine zipper (GILZ), an intracellular protein influencing many signaling pathways, reprograms cancer cell metabolism to promote proliferation. We provided evidence that GILZ overexpression induced a significant increase of mitochondrial oxidative phosphorylation as evidenced by the augmentation in basal respiration, ATP-linked respiration as well as respiratory capacity. Pharmacological inhibition of glucose, glutamine and fatty acid oxidation reduced the activation of GILZ-induced mitochondrial oxidative phosphorylation. At glycolysis level, GILZ-overexpressing cells enhanced the expression of glucose transporters in their plasmatic membrane and showed higher glycolytic reserve. 1H NMR metabolites quantification showed an up-regulation of amino acid biosynthesis. The GILZ-induced metabolic reprograming is present in various cancer cell lines regardless of their driver mutations status and is associated with higher proliferation rates persisting under metabolic stress conditions. Interestingly, high levels of OXPHOS made GILZ-overexpressing cells vulnerable to cell death induced by mitochondrial pro-oxidants. Altogether, these data indicate that GILZ reprograms cancer metabolism towards mitochondrial OXPHOS and sensitizes cancer cells to mitochondria-targeted drugs with pro-oxidant activities.  相似文献   

19.
Bown AW 《Plant physiology》1982,70(3):803-810
Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H+ efflux. Rates varied between 0.12 and 1.99 nanomoles H+ per 106 cells per minute or 3 and 40 nanomoles H+ per milligram chlorophyll per minute. The mean rate of H+ efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H+ efflux from illuminated cells. Bubbling with N2 or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H+ efflux in light or dark. In the absence of aeration, H+ efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO2-free air, rates of H+ efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO2 fixation but not photosynthetic CO2 fixation. It is suggested that H+ efflux is dependent on respiration and dark CO2 fixation, but independent of photosynthesis.  相似文献   

20.
Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K+ channels (mitoKATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca2+-induced mitochondrial permeability transition, and hydrogen peroxide (H2O2) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 μM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (ΔΨ), the phosphorylation rate, and the membrane permeability to H+, K+ and Ca2+ were not affected either. However, sildenafil citrate decreased H2O2 generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O2•−) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 μM do not affect either rat heart mitochondrial bioenergetics or Ca2+-induced mitochondrial permeability transition, but it depresses H2O2 generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.  相似文献   

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