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PubMed1987

31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition.

31P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. Our results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (Pi) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP gamma saturation and were used in combination with ST data to determine Pi consumption rates. 13C NMR and O2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O2 consumption, respectively, under the various metabolic conditions examined. Our results suggest that GAPDH/PGK-catalyzed Pi-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally.

Adenosine Triphosphate↗
PubMed1987

The preventive effect of verapamil on ethanol-induced cardiac depression: phosphorus-31 nuclear magnetic resonance and high-pressure liquid chromatographic studies of hamsters.

Alcoholic depression of left ventricular function was produced in normal hamsters by the administration of increasing concentrations of alcohol in drinking water (up to 50%) for 6 months. The result was assessed by phosphorus-31 nuclear magnetic resonance of isolated perfused hearts and high-pressure liquid chromatography of freeze-clamped tissues. Hemodynamic data and myocardial oxygen consumption were also monitored. Alcoholic hamsters had significantly higher inorganic phosphate and lower ATP levels, while maintaining normal intracellular pH, phosphocreatine, and creatine. Although coronary flow and oxygen consumption were maintained at normal levels, hamsters ingesting 50% ethanol had significantly lower left ventricular developed pressure and dP/dt. Treatment with verapamil during long-term ethanol consumption prevented the development of these metabolic and functional abnormalities. It is hypothesized that alcohol produces membrane abnormalities leading to adverse ion flux, and that these are largely prevented by concurrent administration of verapamil.

Adenosine Triphosphate↗
PubMed1975

Leghaemoglobin and the supply of O2 to nitrogen-fixing root nodule bacteroids: presence of two oxidase systems and ATP production at low free O2 concentration.

Studies of rates of consumption of dissolved O2 by suspensions of bacteroids (Rhizobium japonicum, strain CB1809) from soybean root nodules showed the presence of two different terminal oxidase systems. A high-affinity system, sensitive to inhibition by N-phenylimidazole and by carbon monoxide, was most active when the dissolved O2 was between 0-01 and 0-1 muM. At 1 muM-O2 or higher, this oxidase system had little activity and O2 was consumed largely by a low-affinity system insensitive to these inhibitors. At low concentrations of dissolved O2, bacteroid respiration rates appeared to be diffusion-limited. When purified oxyleghaemoglobin was added to such systems, this restriction was relieved and respiration was maintained to much lower concentrations of free dissolved O2, where nitrogenase activity was greatest. Analysis of reactions which were terminated at various stages during the depletion of O2 from oxyleghaemoglobin showed that at low free O2 concentration, the high-affinity pathway produced up to five times greater bacteroid ATP concentrations than the low-affinity oxidase pathway operating about 1 muM free O2 in the absence of leghaemoglobin. At intermediate free O2 concentrations, occurring during the later stages of deoxygenation of oxymyoglobin, intermediate concentrations of ATP were found in the bacteroids.

Adenosine Triphosphate↗
PubMed1987

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells.

Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Caf) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Caf has been measured using the intracellular Ca-sensitive fluorescent dyes Fura 2 and Quin 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Caf was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Caf was 300-380 nM and, as dye content was increased, Caf progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na-Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Caf, prolonged treatment with ouabain had no effect on Caf despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na-Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Caf. However, exchange-mediated Ca efflux may play a role in Caf regulation when cytosolic calcium is elevated.

Animals↗
PubMed1991

Cytotoxicity of alkylating agents in isolated rat kidney proximal tubular and distal tubular cells.

Patterns of chemical-induced cytotoxicity in different regions of the nephron were studied with freshly isolated proximal tubular and distal tubular cells from rat kidney. Three model alkylating agents, methyl vinyl ketone, allyl alcohol, and N-dimethylnitrosamine, were used as test chemicals. Methyl vinyl ketone and a metabolite of allyl alcohol, acrolein, are Michael acceptors that bind to cellular protein sulfhydryl groups and GSH. N-Dimethylnitrosamine binds to cellular protein and DNA. Lactate dehydrogenase leakage was used to assess irreversible cellular injury. Distal tubular cells were more susceptible than proximal tubular cells to injury produced by methyl vinyl ketone or allyl alcohol while the two cell populations were equally susceptible to injury produced by N-dimethylnitrosamine. Preincubation of both proximal tubular and distal tubular cells with GSH protected them from methyl vinyl ketone- and allyl alcohol-induced cytotoxicity but had no effect on N-dimethylnitrosamine-induced cytotoxicity. Similarly, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, altered cellular GSH status. As with GSH status, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, caused pronounced inhibitory effects on mitochondrial function, as evidenced by ATP depletion and inhibition of cellular oxygen consumption. These results demonstrate that alkylating agents are cytotoxic to both proximal tubular and distal tubular cells, and that interaction with cellular GSH is a factor determining nephron cell type specificity of injury.

1-Propanol↗
PubMed1977

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors.

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.

Adenosine Triphosphatases↗
PubMed1987

1H- and 31P-NMR studies on smooth muscle of bullfrog stomach.

31P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 mumol X g-1 wet wt.), ATP (3.6), inorganic phosphate (Pi, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of Pi. 1H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. 31P saturation transfer studies showed that saturation of the gamma-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T1 value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 mumol X g-1 wet wt. X s-1. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach.

Adenosine Triphosphate↗
PubMed1991

Deoxyadenosine triphosphate acting as an energy-transferring molecule in adenosine deaminase inhibited human erythrocytes.

Deoxyadenosine triphosphate (dATP) is present in adenosine deaminase (ADA)-deficient or ADA-inhibited human red cells and in the red cells of the opossum Didelphis virginiana. In order to investigate the functions of dATP in the red cell, red cells were treated with 2'-deoxycoformycin (dCf), a powerful inhibitor of ADA, and incubated with phosphate, deoxyadenosine and glucose. These red cells in which ATP was almost completely replaced by dATP, had the same shape, lactate production, nucleotide consumption, stability of reduced glutathione, osmotic fragility and cell deformability as red cells containing ATP. Cells merely depleted of ATP showed reduced viability. This indicates that dATP compensates well for the absence of ATP and acts as an energy-transferring molecule to maintain cell viability. These results indicate that the accumulation of dATP or the reduction of ATP is not the cause of the hemolysis observed after dCf administration.

Adenosine Deaminase↗
PubMed2004

Modification of glycolysis affects cell sensitivity to apoptosis induced by oxidative stress and mediated by mitochondria.

The effect of alteration of the glycolytic pathway on cell damage induced by oxidative stress was investigated with dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells that either overexpress cytosolic glycerol-3-phosphate dehydrogenase (CHO/cGPDH cells) or are depleted of the A subunit of lactate dehydrogenase as a result of anti-sense RNA expression (CHO/anti-LDH cells). The extent of oxidative phosphorylation in CHO/anti-LDH and CHO/cGPDH cells was increased and decreased, respectively, relative to that in parental CHO cells, as revealed by measurement of the intracellular content of ATP, the rate of cellular O(2) consumption, the mitochondrial membrane potential (DeltaPsi(m)), and the generation of reactive oxygen species. The sensitivity of these cell lines to cell death induced by the exogenous oxidant tert-butyl hydroperoxide decreased according to the rank order CHO/anti-LDH>CHO>CHO/cGPDH. Exogenous pyruvate markedly increased the sensitivity of CHO/cGPDH cells to oxidant-induced death. The differences among the three cell lines in susceptibility to oxidant-induced death were reflected in the proportion of oxidant-treated cells with a subdiploid DNA content, with a collapsed DeltaPsi(m), and with cytochrome c in the cytosol, indicating that death was mediated by apoptosis. These results demonstrate that the influx of respiratory substrate into mitochondria is an important determinant of cell sensitivity to oxidant-induced apoptosis.

Animals↗
PubMed2004

Prevention of type 1 diabetes: from the view point of beta cell damage.

The hallmark of immune-mediated type 1 diabetes is T cell-mediated destruction of the insulin-producing beta cells in the islets, which results from an imbalance between disease promoting factors and protective elements. The precise mechanisms of beta cell destruction leading to diabetes remain unclear. There are many molecules, including Fas ligand (FasL) and cytokines, such as IL-1, TNF-alpha and IFN-gamma that cause release of other cytokine-mediators that have potential to damage the beta cells. The beta cell-death appears to ultimately be caused by receptor (Fas/FasL)-mediated mechanisms and/or by secretion of cytotoxic molecules (e.g., granzymes, perforin). FasL-mediated beta cell damage might play a role in promoting insulitis and beta cell destruction in autoimmune diabetes in addition to toxic molecules, such as reactive oxygen species (superoxide, hydroxy radical, nitric oxide) or perforin. Furthermore, DNA damage in beta cells leads to poly (ADP-ribose) polymerase-activation which will increase NAD consumption and rapid depletion of NAD compromise ATP production in the cells. Nicotinamide inhibits poly (ADP-ribose) polymerase and reduces nitric oxide accumulation in the NOD pancreas and protect beta cells against radical-induced necrosis. Transgenic mice with beta cell specific overexpression of copper, zinc superoxide dismutase, or thioredoxin are resistant to autoimmune and STZ-induced diabetes. It is apparent that a number of different mechanisms of beta cell destruction are operative in type 1 diabetes. Blockage of multiple pathways, rather than a single pathway, of beta cell-death may, therefore be necessary to fully protect beta cells from destruction and thereby prevent type 1 diabetes.

Animals↗
PubMed2006

Evaluation of sex differences on mitochondrial bioenergetics and apoptosis in mice.

It has been postulated that the differences in longevity observed between organisms of different sexes within a species can be attributed to differences in oxidative stress. It is generally accepted that differences are due to the higher female estrogen levels. However, in some species males live the same or longer despite their lower estrogen values. Therefore, in the present study, we analyze key parameters of mitochondrial bioenergetics, oxidative stress and apoptosis in the B6 (C57Bl/6J) mouse strain. There are no differences in longevity between males and females in this mouse strain, although estrogen levels are higher in females. We did not find any differences in heart, skeletal muscle and liver mitochondrial oxygen consumption (State 3 and State 4) and ATP content between male and female mice. Moreover, mitochondrial H(2)O(2) generation and oxidative stress levels determined by cytosolic protein carbonyls and concentration of 8-hydroxy-2'-deoxyguanosine in mitochondrial DNA were similar in both sexes. In addition, markers of apoptosis (caspase-3, caspase-9 and mono- and oligonucleosomes: the apoptosis index) were not different between male and female mice. These data show that there are no differences in mitochondrial bioenergetics, oxidative stress and apoptosis due to gender in this mouse strain according with the lack of differences in longevity. These results support the Mitochondrial Free Radical Theory of Aging, and indicate that oxidative stress generation independent of estrogen levels determines aging rate.

Animals↗
PubMed2006

Effects of electroconvulsive seizures on Na(+),K(+)-ATPase activity in the rat hippocampus.

Although several advances have occurred concerning the use of electroconvulsive therapy, little progress has been made in understanding the mechanisms underlying its therapeutic or side effects. Na(+),K(+)-ATPase is an important enzyme of central nervous system, responsible for ionic gradient maintenance and consumption of approximately 40-50% of brain ATP. This work was performed in order to determine Na(+),K(+)-ATPase activity after acute and chronic electroconvulsive shock. Results showed an inhibition of Na(+),K(+)-ATPase activity in the hippocampus 48 h, 7, 30, 60 and 90 days after a single electroconvulsive shock. Chronic treatment diminished the enzyme activity in the hippocampus 7 and 30 days after electroconvulsive (ECS) sessions. Our findings demonstrated that Na(+),K(+)-ATPase activity is altered by ECS.

Animals↗
PubMed1999

Three-dimensional redox image of the normal gerbil brain.

Regional differences in the redox ratio were studied in the gerbil brain. Brains were frozen using an in situ funnel-freezing method, and sliced coronally for scanning of mitochondrial redox imaging. The relative local redox ratio of nicotinamide-adenosine dinucleotide to its reduced form was calculated from fluorescence signals of intrinsic fluorochromes, i.e. reduced nicotinamide-adenosine dinucleotide and flavoproteins, using a high resolution fluorometer developed in our laboratory. Twelve consecutive coronal images were obtained from each of 10 gerbils. The mean value of the regional redox ratio in both the cerebral and cerebellar gray matter were found to be significantly lower than that in the cerebral and cerebellar white matter (P < 0.01, Mann-Whitney test). Local differences in the redox ratio were also found among subregions of gray matter. The redox ratio in the globus pallidus was significantly higher than values in other subregions of gray matter (P < 0.01, Mann-Whitney test) We postulate that a high concentration of the reduced form of pyridine nucleotide is maintained to provide redox energy for rapid turnover of ATP in the areas of high energy consumption.

Animals↗
PubMed1995

Thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors.

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

Adenosine Triphosphate↗
PubMed1992

Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells.

Addition of l-[U-(14)C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-(14)C]glutamate, and (c) efflux of [(14)C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H(+)/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H(+)/l-Glu symport may involve both H(+) consumption during 4-aminobutyrate production and ATP-driven H(+) efflux.

Journal Article↗
PubMed1977

Effect of isoproterenol on amino acid levels and protein turnover in skeletal muscle.

The effect of isoproterenol on amino acid concentrations in perfusate and skeletal muscle was studied during a 3-h perfusion of the isolated rat hemicorpus. The beta-adrenergic agonist inhibited the accumulation of alanine, threonine, phenylalanine, tyrosine, lysine, arginine, leucine, and valine and increased the loss of glutamate, aspartate, serine, and isoleucine from the pool of free amino acids in perfusate and muscle. The loss of glutamate was accompanied by a greater accumulation of glutamine. Changes in alanine levels showed the greatest response with a net accumulation of 98 mumol in the controls becoming a net loss of 54 mumol in the experimentals. These changes in amino acid levels were accounted for in part by a 20% decrease in protein degradation. Protein synthesis was not affected by isoproterenol. In addition to an effect on degradation, it appeared that isoproterenol affected amino acid levels by increasing alanine utilization and causing formation of glutamine instead of alanine. Other effects of the drug included increased rates of lactate production, muscle glycogen breakdown, and oxygen consumption, whereas no effects were observed on ATP and creatine phosphate levels. Pyruvate content of muscle was maintained at a higher level in the presence of the drug than in control perfusions.

Alanine↗
PubMed2000

Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines.

We have established two sublines derived from the insulin-secreting mouse pancreatic beta-cell line MIN6, designated m9 and m14. m9 Cells exhibit glucose-induced insulin secretion in a concentration-dependent manner, whereas m14 cells respond poorly to glucose. In m14 cells, glucose consumption and lactate production are enhanced, and ATP production is largely through nonoxidative pathways. Moreover, lactate dehydrogenase activity is increased, and hexokinase replaces glucokinase as a glucose-phosphorylating enzyme. The ATP-sensitive K(+) channel activity and voltage-dependent calcium channel activity in m14 cells are reduced, and the resting membrane potential is significantly higher than in m9 cells. Thus, in contrast to m9, a model for beta-cells with normal insulin response, m14 is a model for beta-cells with impaired glucose-induced insulin secretion. By mRNA differential display of these sublines, we found 10 genes to be expressed at markedly different levels. These newly established MIN6 cell sublines should be useful tools in the analysis of the genetic and molecular basis of impaired glucose-induced insulin secretion.

ATP-Binding Cassette Transporters↗
PubMed2007

Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism.

Obesity-related increase in body fat mass is a risk factor for many diseases, including type 2 diabetes. Controlling adiposity by targeted modulation of adipocyte enzymes could offer an attractive alternative to current dietary approaches. Brown adipose tissue, which is present in rodents but not in adult humans, expresses the mitochondrial uncoupling protein 1 (UCP1) that promotes cellular energy dissipation as heat. Here, we report on the direct metabolic effects of forced UCP1 expression in white adipocytes derived from a murine (3T3-L1) preadipocyte cell line. After stable integration, the ucp1 gene product was continuously expressed during differentiation and reduced the total lipid accumulation by approximately 30% without affecting other adipocyte markers, such as cytosolic glycerol-3-phosphate dehydrogenase activity and leptin production. The expression of UCP1 also decreased glycerol output and increased glucose uptake, lactate output, and the sensitivity of cellular ATP content to nutrient removal. However, oxygen consumption and beta-oxidation were minimally affected. Together, our results suggest that the reduction in intracellular lipid by constitutive expression of UCP1 reflects a downregulation of fat synthesis rather than an upregulation of fatty acid oxidation.

3T3-L1 Cells↗