Stimulation by Adenosine of Adenine Nucleotide Turnover in Isolated Hepatocytes: Evidence for a Futile Cycle Between AMP and Adenosine

1984 ◽  
pp. 475-480
Author(s):  
F. Bontemps ◽  
G. Van den Berghe ◽  
H. G. Hers
Keyword(s):  
Adenine Nucleotide ◽  
Isolated Hepatocytes ◽  
Futile Cycle

scholarly journals Dependence of mitochondrial and cytosolic adenine nucleotides on oxygen partial pressure in isolated hepatocytes. Application of a new rapid high pressure filtration technique for fractionation

1988 ◽  
Vol 250 (3) ◽  
pp. 641-645 ◽  
Author(s):  
H Hummerich ◽  
H de Groot ◽  
T Noll ◽  
S Soboll
Keyword(s):  
High Pressure ◽  
Oxygen Partial Pressure ◽  
Partial Pressure ◽  
Adenine Nucleotide ◽  
Intact Cell ◽  
Adenine Nucleotides ◽  
Strong Dependence ◽  
Isolated Hepatocytes ◽  
Pressure Filtration ◽  
Filtration Technique

By using a new rapid high pressure filtration technique, mitochondrial and cytosolic ATP and ADP contents were determined in isolated hepatocytes at different oxygen partial pressures. At 670 mmHg, subcellular adenine nucleotide contents and ATP/ADP ratios were comparable with values obtained with the digitonin fractionation technique. However at lower oxygen partial pressure ADP appears to be rephosphorylated during digitonin fractionation whereas with high pressure filtration fractionation rephosphorylation of ADP is avoided due to shorter fractionation times. Cytosolic and mitochondrial ATP/ADP ratios decrease if oxygen partial pressure is lowered. However the absolute values of ATP/ADP ratios depend critically on the incubation conditions. Thus incubation of hepatocytes in an oxystat system, where oxygen partial pressure is maintained constant by infusing oxygen-saturated medium and the hepatocyte suspension is continuously stirred, yields much higher subcellular and overall ATP/ADP ratios than incubation in Erlenmeyer flasks gassed with different gas mixtures and shaken in a water bath. This is ascribed to limited diffusion of oxygen from the medium into the cell if the suspension is not mixed thoroughly by stirring. The strong dependence of subcellular ATP/ADP ratios on incubation conditions indicates that oxygen may be one rate-controlling factor for oxidative phosphorylation in the intact cell.

Model of β-cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables

1998 ◽  
Vol 274 (4) ◽  
pp. C1174-C1184 ◽  
Author(s):  
Gerhard Magnus ◽  
Joel Keizer
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Adenine Nucleotide ◽  
Tricarboxylic Acid Cycle ◽  
Calcium Handling ◽  
Β Cell ◽  
Futile Cycle ◽  
Cyclic Changes

In the preceding article [ Am. J. Physiol. 274 ( Cell Physiol. 43): C1158–C1173, 1998], we describe the development of a kinetic model for the interaction of mitochondrial Ca2+ handling and electrical activity in the pancreatic β-cell. Here we describe further results of those simulations, focusing on mitochondrial variables, the rate of respiration, and fluxes of metabolic intermediates as a function of d-glucose concentration. Our simulations predict relatively smooth increases of O2consumption, adenine nucleotide transport, oxidative phosphorylation, and ATP production by the tricarboxylic acid cycle asd-glucose concentrations are increased from basal to 20 mM. On the other hand, we find that the active fraction of pyruvate dehydrogenase saturates, due to increases in matrix Ca2+, near the onset of bursting electrical activity and that the NADH/NAD+ ratio in the mitochondria increases by roughly an order of magnitude as glucose concentrations are increased. The mitochondrial ATP/ADP ratio increases by factor of <2 between thed-glucose threshold for bursting and continuous spiking. According to our simulations, relatively small changes in mitochondrial membrane potential (∼1 mV) caused by uptake of Ca2+ are sufficient to alter the cytoplasmic ATP/ADP ratio and influence ATP-sensitive K+ channels in the plasma membrane. In the simulations, these cyclic changes in the mitochondrial membrane potential are due to synchronization of futile cycle of Ca2+ from the cytoplasm through mitochondria via Ca2+ uniporters and Na+/Ca2+exchange. Our simulations predict steady mitochondrial Ca2+concentrations on the order of 0.1 μM at low glucose concentrations that become oscillatory with an amplitude on the order of 0.5 μM during bursting. Abrupt increases in mitochondrial Ca2+concentration >5 μM may occur during continuous electrical activity.

scholarly journals Is the adenine nucleotide translocator rate-limiting for oxidative phosphorylation?

1978 ◽  
Vol 172 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Marion Stubbs ◽  
Pierre V. Vignais ◽  
Hans A. Krebs
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Adenine Nucleotide ◽  
Phosphoglycerate Kinase ◽  
Isolated Hepatocytes ◽  
Urea Synthesis ◽  
Intermediary Metabolites ◽  
Glucose Synthesis ◽  
High Concentrations ◽  
K Concentration ◽  
Rate Limiting

1. The effects of atractyloside and carboxyatractyloside (between 5 and 40μm) on O2 uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K+ concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]×[Pi] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K+]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett.74, 50–54] assume. This point is discussed.

Purine metabolism in isolated rat hepatocytes

1977 ◽  
Vol 55 (11) ◽  
pp. 1134-1139 ◽  
Author(s):  
Camilla M. Smith ◽  
Liisa M. Rovamo ◽  
Martti P. Kekomäki ◽  
Kari O. Raivio
Keyword(s):  
Cell Function ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Purine Metabolism ◽  
Nucleotide Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Collagenase Perfusion ◽  
Adenine Nucleotide Pool

The metabolism of adenine, hypoxanthine, guanine, and adenosine was studied in rat liver cell suspensions, prepared by collagenase perfusion. Oxygen supply was a critical variable in the preparation and subsequent incubation of the cells, as judged on the basis of the ratio of radioactivity in ATP to that in ADP after incubation with [14C]adenine. This ratio is suggested as an additional criterion of cell function. Adenine nucleotides synthesized from [14C]adenine were slowly catabolized to allantoin, with little incorporation of radioactivity into other purine compounds. [14C]Adenine is thus suitable for prelabelling the adenine nucleotide pool. [14C]Guanine and [14C]hypoxanthine were rapidly catabolized to allantoin, whereas nucleotide synthesis was low. [14C]Adenosine was initially phosphorylated and deaminated at about equal rates, but with continued incubation catabolic products predominated. Isolated hepatocytes were found suitable for studies of purine metabolism, in which the liver has important functions for the whole organism.

Mitochondrial transmembrane potential and pH gradient during anoxia

1987 ◽  
Vol 252 (4) ◽  
pp. C349-C355 ◽  
Author(s):  
B. S. Andersson ◽  
T. Y. Aw ◽  
D. P. Jones
Keyword(s):  
Transmembrane Potential ◽  
Adenine Nucleotide ◽  
Electron Flow ◽  
Atp Synthesis ◽  
Isolated Hepatocytes ◽  
Mitochondrial Transmembrane Potential ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Ion Distribution ◽  
Cytosolic Ph

The effect of anoxia on the mitochondrial transmembrane potential and pH gradient was studied in a preparation of isolated hepatocytes. Transmembrane potential (delta psi) was calculated from the distribution of triphenylmethylphosphonium between the mitochondrial, cytosolic, and extracellular compartments, which were separated by digitonin fractionation and centrifugation. Mitochondrial and cytosolic pH values were calculated from the distribution of the weak acid, dimethadione, which was determined similarly. After 30 min anoxia, the magnitude of mitochondrial delta psi was decreased from -163 to -133 mV and the delta pH (mitochondria vs. cytoplasm) was essentially unchanged (aerobic, 0.78 +/- 0.08; anaerobic, 0.76 +/- 0.11). Thus the protonmotive force (delta p = delta psi-Z delta pH), is largely retained even in the absence of electron flow and ATP synthesis. Inhibitors of the ATP synthase (oligomycin), mitochondrial adenine nucleotide carrier (atractyloside), and glycolytic pathway (2-deoxy-D-glucose) do not affect the ability of the cell to maintain delta psi during anoxia. Therefore, the results indicate that retention of the protonmotive force is not due to utilization of ATP produced by glycolysis and suggest that mechanisms exist to preserve ion distribution during anoxia.

Regulation of the mitochondrial ATP-Mg/Pi carrier in isolated hepatocytes

1993 ◽  
Vol 264 (3) ◽  
pp. C663-C670 ◽  
Author(s):  
D. T. Dransfield ◽  
J. R. Aprille
Keyword(s):  
Subcellular Distribution ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Isolated Hepatocytes ◽  
Cellular Regulation ◽  
Intact Cells ◽  
Nucleotide Content ◽  
Dose Dependent

This study investigated the cellular regulation of net adenine nucleotide movements between the cytoplasm and mitochondria in intact cells. Such movements are presumed to occur primarily by ATP-Mg exchange with Pi via the mitochondrial ATP-Mg/Pi carrier. Vasopressin, A23187, and thapsigargin all elevate intracellular free [Ca2+] and all caused dose-dependent increases in the mitochondrial adenine nucleotide content (29, 63, and 39%, respectively). Phorbol 12-myristate 13-acetate had no effect. The effect of vasopressin was abolished when cytoplasmic [ATP] was decreased (by 43%) and [Pi] was increased (3-fold) by addition of carboxyatractyloside. The effect of thapsigargin was abolished by addition of xylulose to deplete cytoplasmic [ATP] (by 50%) and [Pi] (> 4-fold). The results indicate that in intact cells Ca2+ activates the mitochondrial ATP-Mg/Pi carrier to enable changes in the subcellular distribution of adenine nucleotides and that the relative [ATP] and [Pi] gradients govern the direction and magnitude of net adenine nucleotide movements between the cytoplasm and mitochondria.

BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes

1998 ◽  
Vol 275 (4) ◽  
pp. H1260-H1266 ◽  
Author(s):  
Mary T. Stapleton ◽  
Claudia M. Fuchsbauer ◽  
Ashley P. Allshire
Keyword(s):  
Atp Hydrolysis ◽  
Adenine Nucleotide ◽  
Atp Depletion ◽  
Metabolic Inhibitors ◽  
Nucleotide Exchange ◽  
Futile Cycle ◽  
Intact Cells ◽  
Rat Cardiomyocytes ◽  
Permeabilized Cells ◽  
Adult Rat

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10–50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

1996 ◽  
Vol 54 ◽  
pp. 966-967
Author(s):  
C.A. Mannella ◽  
K.F. Buttle ◽  
K.A. O‘Farrell ◽  
A. Leith ◽  
M. Marko
Keyword(s):  
Liver Mitochondrion ◽  
Adenine Nucleotide ◽  
Protein Complexes ◽  
Mitochondrial Membranes ◽  
Electron Microscopic ◽  
Contact Sites ◽  
Transmission Electron ◽  
Precursor Proteins ◽  
Membrane Contacts ◽  
And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

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