Total ATP Content Research Articles

Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS)

The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight.

Opposite effects of d-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells

d-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM d-glucose. Even in the absence of d-glucose, d-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of d-fructose could be attributed to an increase in the ATP content of islet cells. After 30–60 min incubation in the presence of d-glucose and/or d-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). d-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM d-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, d-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of d-glucose and/or d-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either 86Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.

ATP dependence of the non-specific ion channel in Torpedo synaptic vesicles

Synaptic vesicles of Torpedo electromotor neurons contain a high amount of ATP. The concentration of total ATP is around 120 mM, whereas the free [ATP] is about 5-6 mM. We examined the effect of intravesicular ATP on the non-specific ion channel in Torpedo-fused synaptic vesicles. It was found that this channel is closed when the ATP concentration is above 2 mM, but it is very frequently open at lower ATP concentrations. Unmasking this ion channel at a low ATP concentration may be significant for post-fusion control of transmitter release by the 'kiss and run' mechanism in normal conditions, while during metabolic stress it may underlie dissipation of important gradients across the vesicle membrane.

Propionyl‐L‐carnitine prevents The Progression of Cisplatin‐Induced Cardiomyopathy in a Carnitine‐Depleted Rat Model

This study is to investigate whether endogenous carnitine depletion and/or carnitine deficiency is a risk factor during development of cisplatin (CDDP)-induced cardiomyopathy and if so, whether carnitine supplementation by propionyl-L-carnitine (PLC) could offer protection against this toxicity. A total of 60 adult male rats were divided into 6 groups. The first three groups were injected intraperitoneally with normal saline, PLC (500 mg kg-1), and D-carnitine (500 mg kg-1) respectively, for 10 successive days. The 4th, 5th, and 6th groups were injected intraperitoneally with the same doses of normal saline, PLC and D-carnitine, respectively for 5 successive days before and after a single dose of CDDP (7 mg kg-1). Six days after CDDP treatment, animals were sacrificed, serum as well as hearts were isolated and analyzed. In the carnitine-depleted rat model, CDDP induced dramatic increase in serum cardiomyopathy enzymatic indices, CK-MB and LDH, as well as progressive reduction in total carnitine and ATP content in cardiac tissue. Interestingly, PLC supplementation attenuated CDDP-induced cardiomyopathy manifested by normalizing the increase of serum CK-MB and LDH, TBARS and NOx, and the decrease in total carnitine, GSH and ATP content in cardiac tissues. In conclusion, data from this study suggest for the first time that carnitine deficiency and oxidative stress are risk factors and should be viewed as mechanisms during development of CDDP-related cardiomyopathy and that carnitine supplementation, using PLC, prevents the progression of CDDP-induced cardiotoxicity.

Protective Effect of Reamberin on Functional Activity of Mitochondria during Skin Ischemia

Reamberin in a dose of 25 mg/kg (succinate concentration) was injected intravenously for 3 days starting from the 1st hour after skin ischemia modeling. This treatment decreased activities of lactate dehydrogenase, aspartate transaminase, and creatine phosphokinase in skin homogenates by 1.6 times, 19%, and 51.3%, respectively. The index of cytolysis decreased by 18%. Reamberin had an energotropic effect, which manifested in an increase in the total ATP content and concentration of creatine phosphate (by 16 and 10%, respectively). After administration of Reamberin, activity of the succinate-ubiquinone reductase system increased by 17%. Under these conditions succinate dehydrogenase activity exceeded the normal by 21%. Reamberin had no effect on the mitochondrial NADH-ubiquinone reductase system in dermal cells during skin ischemia. Superoxide dismutase activity in the area of necrosis increased to the control level on day 3 of treatment with Reamberin. Activities of catalase and glutathione peroxidase increased by 13 and 19%, respectively. Our results indicate that the course of intravenous treatment with Reamberin for 3 days contributes to an increase in reserve capacities of the antioxidant protection system and produces a protective effect during skin ischemia.

In Vitro Screening of Exogenous Factors for Human Neural Stem/Progenitor Cell Proliferation Using Measurement of Total ATP Content in Viable Cells.

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients ( n = 9) and healthy controls ( n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 ± 0.95 nmol/mg versus 0.09 ± 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 ± 0.83 nmol/mg versus 0.29 ± 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.

Activation of Human Meiosis-specific Recombinase Dmc1 by Ca2+

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.

Inhibition of Mitochondrial Na+-Ca2+ Exchange Restores Agonist-induced ATP Production and Ca2+ Handling in Human Complex I Deficiency

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.

ATP - bioluminescence as a technique to evaluate the microbiological quality of water in food industry

ATP-bioluminescence was used to evaluate the microbiological quality of water samples collected from the water supply, the water treatment system and from a dairy plant, including ammonia-cooling water and industrial water. For industrial water, there was relation between the ATP-bioluminescence technique and microbial count. There were no differences (p&gt;0.05) between water supply and ammonia-cooling water samples for total and free ATP concentrations nor for the microbial counts. Different microbial ATP concentrations were found for these water samples. The results suggested that the physical chemical quality of ammonia cooling water decreased the RLU measurements slightly. It could be concluded that the total ATP concentration was the most effective technique to evaluate the microbiological quality of water used in the food indsutry by ATP-bioluminescence.

Oligonucleotide Microarray Analysis Reveals PDX1 as an Essential Regulator of Mitochondrial Metabolism in Rat Islets

Mutations in the transcription factor IPF1/PDX1 have been associated with type 2 diabetes. To elucidate beta-cell dysfunction, PDX1 was suppressed by transduction of rat islets with an adenoviral construct encoding a dominant negative form of PDX1. After 2 days, there was a marked inhibition of insulin secretion in response to glucose, leucine, and arginine. Increasing cAMP levels with forskolin and isobutylmethylxanthine restored glucose-stimulated insulin secretion, indicating normal capacity for exocytosis. To identify molecular targets implicated in the altered metabolism secretion coupling, DNA microarray analysis was performed on PDX1-deficient and control islets. Of the 2640 detected transcripts, 70 were up-regulated and 56 were down-regulated. Transcripts were subdivided into 12 clusters; the most prevalent were associated with metabolism. Quantitative reverse transcriptase-PCR confirmed increases in succinate dehydrogenase and ATP synthase mRNAs as well as pyruvate carboxylase and the transcript for the malate shuttle. In parallel there was a 50% reduction in mRNA levels for the mitochondrially encoded nd1 gene, a subunit of the NADH dehydrogenase comprising complex I of the mitochondrial respiratory chain. As a consequence, total cellular ATP concentration was drastically decreased by 75%, and glucose failed to augment cytosolic ATP, explaining the blunted glucose-stimulated insulin secretion. Rotenone, an inhibitor of complex I, mimicked this effect. Surprisingly, TFAM, a nuclear-encoded transcription factor important for sustaining expression of mitochondrial genes, was down-regulated in islets expressing DN79PDX1. In conclusion, loss of PDX1 function alters expression of mitochondrially encoded genes through regulation of TFAM leading to impaired insulin secretion.

Sex-specific changes in platelet aggregation and secretion with sexual maturity in pigs.

Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2-3 mo) and sexually mature (adult; 5-6 mo) male and female pigs (n=8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 microM) and collagen (6 microg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.

Some processes of energy saving and expenditure occurring during ethanol perfusion in the isolated liver of fed rats; a Nuclear Magnetic Resonance study.

BackgroundIn the isolated liver of fed rats, a 10 mM ethanol perfusion rapidly induced a rapid 25% decrease in the total ATP content, the new steady state resulting from both synthesis and consumption. The in situ rate of mitochondrial ATP synthesis without activation of the respiration was increased by 27%, implying an increased energy demand. An attempt to identify the ethanol-induced ATP-consuming pathways was performed using 31P and 13C Nuclear Magnetic Resonance.ResultsEthanol (i) transiently increased sn-glycerol-3-phosphate formation whereas glycogenolysis was continuously maintained; (ii) decreased the glycolytic ATP supply and (iii) diminished the intracellular pH in a dose-dependent manner in a slight extend. Although the cytosolic oxidation of ethanol largely generated H+ (and NADH), intracellular pHi was maintained by (i) the large and passive excretion of cellular acetic acid arising from ethanol oxidation (evidenced by exogenous acetate administration), without energetic cost or (ii) proton extrusion via the Na+-HCO3- symport (implying the indirect activation of the Na+-K+-ATPase pump and thus an energy use), demonstrated during the addition of their specific inhibitors SITS and ouabaïn, respectively.ConclusionVarious cellular mechanisms diminish the cytosolic concentration of H+ and NADH produced by ethanol oxidation, such as (i) the large but transient contribution of the dihydroxyacetone phosphate / sn-glycerol-3-phosphate shuttle between cytosol and mitochondria, mainly implicated in the redox state and (ii) the major participation of acetic acid in passive proton extrusion out of the cell. These processes are not ATP-consuming and the latter is a cellular way to save some energy. Their starting in conjunction with the increase in mitochondrial ATP synthesis in ethanol-perfused whole liver was however insufficient to alleviate either the inhibition of glycolytic ATP synthesis and/or the implication of Na+-HCO3- symport and Na+-K+-ATPase in the pHi homeostasis, energy-consuming carriers.

Solid Phase Treatment of an Aged Soil Contaminated by Polycyclic Aromatic Hydrocarbons

Laboratory scale tests were carried out in order to evaluate the removal efficiency of polyaromatic hydrocarbons (PAHs) during the different biological treatments of a Manufacturing Gas Plant site aged soil, heavily contaminated by high molecular weight compounds. Biodegradation studies were carried out at nearly 25°C in solid phase reactors. Three tests were performed, over a period of 100 days for each test. In the first test (P1-bioaugmentated), soil was mixed with wood chips and urea at the start of the treatment and after six weeks from the beginning of the test was also periodically inoculated (at 42, 54, 69, 82, and 96 days) with selected consortia of autochthonous PAH-degrading bacteria. The second test (P2-biostimulated) was performed similarly to the previous one, but without any inoculations. In the third test (P3-control) only soil was introduced. All systems were aerated daily and humidified at the occurrence. PAH concentration, total cultivable heterotrophs, PAH-degrading bacteria, mycetes, pH, ATP concentration, and enzymatic activities were monitored every two weeks during the treatments. Tests showed that nearly 50% of light (three rings) PAHs, 35% of benzo-PAHs and 40% of the total PAHs could be removed in the reactor P2 following 100 days of treatment. Lower removal efficiency could be observed for light PAHs (28%) in the inoculated reactor (P1) at the end of the treatment; comparable abatements were obtained for benzo- and total PAHs. In the reactor P3 (control), the concentration of all polyaromatic hydrocarbons was nearly always constant, suggesting that the physical losses were negligible during the solid phase treatments. Therefore the C to N ratio balance resulted to be the key factor in promoting the biodegradation process of all PAHs.

Relationship between the host cell mitochondria and the parasitophorous vacuole in cells infected with Encephalitozoon microsporidia.

Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.

Slowing effects of Mg2+ on contractile kinetics of skinned preparations of rat hearts depending on myosin heavy chain isoform content.

The effects of changes in Mg2+ concentration on the kinetics of stretch activation were investigated on skinned rat heart preparations under maximal Ca2+ activation. Muscle strips of hyper- and hypothyroid rat hearts were investigated at 0.5 and 1 mM free Mg2+; the total ATP concentration was 8 mM which resulted in saturating MgATP2- concentrations above 5 mM. Preparations containing exclusively the cardiac alpha-myosin heavy chain (hyper- and hypothyroid atria, hyperthyroid ventricles) showed an acceleration of the kinetics of stretch activation by a factor of about 1.5 (P<0.01, paired t-test) when free Mg2+ was decreased from 1 to 0.5 mM. Conversely, preparations containing exclusively the beta-myosin heavy chain isoform showed only a small acceleration by a factor of 1.05 (P<0.05, paired t-test) under the same conditions. The fact that the Mg2+ sensitivity was dependent on the myosin heavy chain isoform excludes the possibility that Mg2+ exhibits only unspecific effects on contractile proteins. Several hypotheses for explaining the observed Mg2+ effects are discussed. The conditions used in our experiments might be close to the physiological situation and, thus, changes of Mg2+ concentration must be considered as possible factors modulating the contractile kinetics especially of atrial muscle tissue.

Control of neurotransmitter release by an internal gel matrix in synaptic vesicles.

Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

Adenine Nucleotide and Creatine Phosphate Pool in Adult and Old Rat Heart during Immobilization Stress

Background: Disturbances in the heart energy provision is important in the stress-induced injury of myocardium. In order to learn the causes of age-dependent differences in myocardial sensitivity to stress, we determined the level of adenylates, creatine and creatine phosphate in adult and old rat heart during an immobilization stress. Methods: Fifty male Wistar rats were used to study the myocardial adenine nucleotides, creatine and creatine phosphate pool and creatine kinase activity during an immobilization period. Results: The concentration of adenine nucleotides in old and adult rat hearts was similar. The total concentration of adenylate pool, ATP and ADP in the heart of both age groups was reduced during stress. However, in old rats under stress the concentration of ATP in a less measure and ADP in a greater measure is decreased against that observed in adult animals. The level of inorganic phosphate in old rat heart remains the same as in adult rats. Conclusion: The creatine kinase system may be important in stabilizing the ATP level in myocardium of adult rats during stress. The role of this system in heart energy metabolism during stress is decreased in old rats. Disturbances of isoenzyme creatine kinase activity are responsible for these disorders.

Modulation of islet ATP content by inhibition or stimulation of the Na +/K + pump

High (30 mM) K +, known to cause β-cell membrane depolarisation, significantly decreased the islet total ATP content, supporting the view that β-cell membrane depolarisation can activate the ATP-consuming Na +/K + pump. Ouabain (1 mM) did not change the islet ATP content after 5–15 min of incubation in the absence or presence of 3 mM glucose but reduced it after 30 min, and in the presence of 20 mM glucose, the reduction by ouabain occurred already after 15 min. Incubation of islets with ouabain for 60 min decreased the islet ATP content in the presence of 3, 10 or 20 mM glucose or 30 mM K +. Also, the islet glucose oxidation rate was decreased by ouabain. When K + deficiency was used to inhibit the Na +/K + pump, no change in ATP content was observed irrespective of glucose concentration, although K + deficiency caused a slight inhibition of the glucose oxidation rate. Diazoxide reduced the islet glucose oxidation rate and increased the islet ATP content in the presence of 20 mM glucose. There may exist a feedback mechanism decreasing the flow of glucose metabolism in response to reduced ATP consumption by the Na +/K + pump.

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C.

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.