Absolute Concentration Of ATP Research Articles

АТР-чутливі K+-канали м’язових клітин:

ATP-sensitive K(+)-channels of plasma membranes belong to the inward rectifier potassium channels type. They are involved in coupling of electrical activity of muscle cell with its metabolic state. These channels are heterooctameric and consist of two types of subunits: four poreforming (Kir 6.x) and four regulatory (SUR, sulfonylurea receptor). The Kir subunits contain highly selective K+ filter and provide for high-velocity K+ currents. The SUR subunits contain binding sites for activators and blockers and have metabolic sensor, which enables channel activation under conditions of metabolic stress. ATP blocks K+ currents through the ATP-sensitive K(+)-channels in the most types of muscle cells. However, functional activity of these channels does not depend on absolute concentration of ATP but on the ATP/ADP ratio and presence of Mg2+. Physiologically active substances, such as phosphatidylinositol bisphosphate and fatty acid esters can regulate the activity of these structures in muscle cells. Activation of these channels under ischemic conditions underlies their cytoprotective action, which results in prevention of Ca2+ overload in cytosol. In contrast to ATP-sensitive K(+)-channels of plasma membranes, the data regarding the structure and function of ATP-sensitive K(+)-channels of mitochondrial membrane are contradictory. Pore-forming subunits of this channel have not been firmly identified yet. ATP-sensitive K+ transport through the mitochondrial membrane is easily tested by different methods, which are briefly reviewed in this paper. Interaction of mitoK(ATP) with physiological and pharmacological ligands is discussed as well.

Real-time luminescence imaging of cellular ATP release

Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin–luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin–luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility.

Non-Invasive in Vivo Determination of the Absolute ATP Concentration in the Rat Liver by 31P NMR Spectroscopy

The concentration of ATP in the rat liver was determined in vivo by means of surface coil 31P NMR spectroscopy. The double standard method was used in order to achieve absolute quantification of ATP through its 31P β-resonance. Contamination of the 31P NMR liver spectra by contributions from abdominal muscle tissue was minimized by applying the FROGS presaturation sequence. Difficulties inherent to the B1 inhomogeneity of surface coils were overcome by the use of adiabatic half passage sech/tanh RF pulses. While in the normal rat liver the mean ATP concentration was 3.39 ± 0.31 mM, fibrotic rat livers exhibited a significantly lower average ATP concentration of 2.29 ± 0.31 mM.

What determines the intracellular ATP concentration.

Analysis is made of the mechanisms that control the intracellular ATP level. The balance between energy production and expenditure determines the energy charge of the cell and the ratio of [ATP] to the adenylate pool. The absolute ATP concentration is determined by the adenylate pool, which, in its turn, depends on the balance between the rates of AMP synthesis and degradation. Experimental data are discussed that demonstrate an increase in the adenylate pool in response to activation of energy-consuming processes. A hypothesis is proposed according to which variation in the adenylate pool and absolute ATP concentration affords a cell the possibility of additional control over processes fulfillinguseful work. A mechanism involved in this regulation is described using human erythrocytes as an example. The hypothesis explains why different metabolic pathways (protein and DNA syntheses, polysaccharide synthesis, and lipid synthesis) use different trinucleotides (GTP, UTP, and CTP, respectively) as an energy source. This allows the cell to independently control these metabolic processes by varying the individual nucleotide pools.

A comparison of effect of temperature on phosphorus metabolites, pH and Mg2+ in human and ground squirrel red cells.

1. 31P NMR spectra were obtained at temperatures ranging from 2 to 30 degrees C from freshly drawn human (cold-sensitive) and ground squirrel (cold-tolerant) red cells. The concentration of ATP was also determined by luciferin-luciferase assay over the same temperature range. 2. The concentration of ATP as determined by NMR or by the luciferin-luciferase assay did not change with temperature in either species. The absolute concentration of ATP in human cells determined by NMR was not significantly different from the total ATP determined enzymatically. 3. The concentration of 2,3-diphosphoglycerate was higher and that of pyridine nucleotides lower in human than in ground squirrel red cells. This species difference was independent of temperature. 4. Intracellular pH, as determined from the positions of the NMR peaks of 2- and 3-phosphates of diphosphoglycerate, became more alkaline as the temperature was lowered. 5. Free intracellular magnesium, determined from the difference in the positions of the peaks for alpha- and beta-phosphorus of ATP, increased in the ground squirrel red cells and decreased in the human red cells with cooling from 30 to 2 degrees C. Total magnesium, as determined by atomic emission spectroscopy, did not change with temperature in red cells of either species. 6. The intensities of all phosphorus metabolite signals from the ground squirrel cells increased with decreasing temperature, while those from the human cells were unaffected. Since chemical shift anisotropy in the presence of magnesium is a powerful spin-lattice relaxation mechanism for phosphates, this is additional evidence for the temperature dependence of free magnesium concentration in the ground squirrel cells. 7. We conclude that there is no difference in phosphorus metabolites or intracellular pH which could account for the differential cold sensitivity in human and ground squirrel red cells. We suggest that, in the cold-tolerant red cells from the ground squirrel, magnesium is released from binding sites as the temperature is lowered. The change in free intracellular Mg2+ may account at least in part for the unusually low temperature sensitivity of the Na(+)-K+ pump in the red cells of this species.

Adenine Nucleotide Depletion in Human Muscle During Exercise: Causality and Significance of AMP Deamination

The predominant route for adenine nucleotide catabolism in skeletal muscle is deamination of AMP to inosine monophosphate (IMP) and ammonia (NH3). Deamination of AMP is enhanced during exercise when the capacity to rephosphorylate ADP is impaired. Thus, in human muscle the formation of IMP (NH3) during exercise is augmented under the following conditions (1) at high intensities, (2) during beta-adrenoceptor blockade, (3) during hypoxia, (4) after detraining, and (5) at low glycogen levels. The formation of IMP is related to the metabolic stress (as indicated by the degree of phosphocreatine breakdown and lactate accumulation), the rate of ATP turnover, and the fiber type composition. During maximal exercise at 100% of VO2max or sustained isometric contractions to fatigue, about 15% of the adenine nucleotide (AN) pool is degraded through deamination of AMP to IMP. It is suggested that the stimulus for increased AMP deamination is increased transient levels of ADP and AMP in the contracting muscle fiber. Deamination of AMP to IMP and NH3 provides a sink for ADP, whereby the ATP/ADP ratio and the phosphorylation potential are kept high, which may be essential for the continuation of the contraction process. This implies that the relative levels of the adenine nucleotides are more important for maintenance of adequate cellular function than the absolute concentration of ATP.

Effects of insulin and dexamethasone on adenine nucleotide levels in cultured hepatocytes from adult rat.

Insulin and dexamethasone, usually added to culture media, play an important role in maintaining the survival of functional hepatocytes. Adenine nucleotide concentrations and energy charge values of cultured hepatocytes were determined to investigate the relationship between the beneficial effects of these hormones and the energy status of the cells. The results indicate that insulin and dexamethasone are essential in maintaining the metabolic competence of cultured hepatocytes and that this correlates with the absolute concentration of ATP rather than with the energy charge.

Variation in adenylate energy charge and phosphoadenylate pool size in estuarine organisms after an oil spill.

Adenylate energy charge (AEC) is the proportion of the total phosphoadenylate pool charged with high-energy bonds. AEC values vary between zero and one by definition. Since AEC can be measured in any organism, decreases might be a universal measure of sublethal environmental stress. In some organisms which maintain high AEC while withstanding natural or anthropogenic stress, the absolute concentration of ATP and the total phosphoadenylate pool (TPP) decrease proportionally. However, in certain organisms the TPP shows dramatic natural fluctuations unrelated to pollution or stress. On 28 June 1983, a tanker spilled approximately 42,000 gallons of number6 diesel oil in the Cape Fear River, North Carolina, USA. Oil covered the tidal marshes on the east side of the river and provided an opportunity to determine if either the AEC or TPP in a variety of organisms would respond to this stress. Five test species were examined as long as one year after the spill. AEC and TPP values of the organisms were compared between contaminated and uncontaminated sites at all seasons. This is the first investigation to monitor AEC in a number of taxonomically distinct estuarine species during an extended period after an oil spill.

Biochemical and Morphological Studies on Human Kidneys Preserved for Transplantation

Seven human kidneys that had been preserved for transplantation by pulsatile perfusion were studied to correlate the biochemical data with morphologic changes. Metabolite concentrations in mumol/g wet tissue were ATP = 0.26; ADP = 0.34; AMP = 0.45; lactate = 15.21; pyruvate = 0.23; 3-phosphoglycerate = 0.05; fructose-1,6-bisphosphate = 0.06; and hexose-6-phosphate = 0.03. Enzyme activities in mumol/min . mg protein found in the microsomal fraction were alkaline phosphatase = 0.049 and gamma-glutamyl transpeptidase = 0.844. Morphologically, none of the kidneys showed irreversible cell injury in the renal tubules, but some glomeruli showed areas where the endothelial cells appeared stripped off of the capillary basement membranes, indicating possible perfusion injury. The data suggest that it is the resynthesizing ability, as opposed to the absolute concentration of ATP, which determines the recovery and the subsequent viability of the tissue.

Metabolic studies of postischemic acute renal failure in the rat

Postischemic acute renal failure was induced by 1 hr of clamping of the renal vasculature. Adenine nucleotide (ATP, ADP, AMP) and lactate (Lac) levels were measured after 0, 0.25, 1, 6, 24, and 48 hr of reflow to determine the time necessary for recovery to control levels. After 1 hr of ischemia with no reflow, [ATP] was 18% and [Lac] was 10-fold control levels. Control levels were restored after 24 hr of reflow. Variable ischemic times (5, 15, 30, 60, 90, and 120 min) followed by (1) no reflow or (2) 24 hr of reflow were also studied. [ATP] decreased to 25 and 13% of controls after 5 and 120 min of ischemia, respectively, and [Lac] increased to 5- and 13-fold controls after 5 and 120 min. Five to ninety minutes of ischemia followed by 24 hr of reflow resulted in a trend toward restoration of ATP and Lac levels; whereas, 120 min of ischemia followed by 24 hr of reflow resulted in death. The results indicate that: (1) In vivo ischemia results in a drastic and rapid shift in the ATP-ADP-AMP equilibrium; (2) the absolute concentration of ATP is not a reliable criterion of cell viability, but the ability to resynthesize ATP may be determinant in the reversibility of the lesion; (3) 1 hr of ischemia is reversible with respect to restoration of [ATP] and [Lac], but 24 hr of reflow are needed for restoration; and (4) ischemia for 90 min results in a metabolic derangement which is partially reversible in that metabolite levels are partially restored after 24 hr of reflow. However, 90 min of vascular clamping is not functionally reversible since the majority of animals exhibit severe azotemia and do not survive.

Biochemical and morphological studies on human kidneys preserved for transplantation.

Seven human kidneys that had been preserved for transplantation by pulsatile perfusion were studied to correlate the biochemical data with morphologic changes. Metabolite concentrations in mumol/g wet tissue were ATP = 0.26; ADP = 0.34; AMP = 0.45; lactate = 15.21; pyruvate = 0.23; 3-phosphoglycerate = 0.05; fructose-1,6-bisphosphate = 0.06; and hexose-6-phosphate = 0.03. Enzyme activities in mumol/min . mg protein found in the microsomal fraction were alkaline phosphatase = 0.049 and gamma-glutamyl transpeptidase = 0.844. Morphologically, none of the kidneys showed irreversible cell injury in the renal tubules, but some glomeruli showed areas where the endothelial cells appeared stripped off of the capillary basement membranes, indicating possible perfusion injury. The data suggest that it is the resynthesizing ability, as opposed to the absolute concentration of ATP, which determines the recovery and the subsequent viability of the tissue.

Investigation of the primary ionization of the terminal phosphate group in adenosine-5?-triphosphoric acid by potentiometric titration

1. It has been established by potentiometric titration that only one proton is added to the terminalγ-phosphate group when ATP (0.05–0.25 M) is protonated at pH 3-1.3. 2. At a given pH the fraction of the protonated form depends on the absolute concentration of ATP. The results have been quantitatively interpreted with consideration of the dimerization of ATP. The equilibrium constants between the monomeric forms A2− and A− and for the formation of the dinier A2−A2− have been determined at 25‡C. 3. The protonation of the terminal phosphate promotes the destruction of the dimer.

Inhibition of lymphocyte function by 9-deazaadenosine

9-Deazaadenosine (c 9Ado), a novel C-nucleoside, has been found to inhibit lymphocytemediated cytolysis (LMC) in a time-dependent manner. c 9Ado inhibited LMC by 50% at concentrations of 10 and 0.07 μM after drug-pretreatment periods of 3 and 22 hr, respectively, although a 1-hr pretreatment of cytolytic lymphocytes with 100 μM c 9Ado had no effect upon this lymphocyte function. c 9Ado was metabolized rapidly and extensively to 9-deazaadenosine 5'-triphosphate (c 9ATP) both by mouse cytolytic lymphocytes and by human erythrocytes. Adenosine kinase purified from rabbit liver phosphorylated c 9Ado with a K m of 200 μM and a V max of 8% that for adenosine. The metabolic buildup of c 9ATP in lymphocytes was accompanied by a large, time-dependent decrease in cellular ATP and by smaller percentage decreases in CTP, UTP and GTP. Among other biochemical effects examined, c 9Ado was found to cause a decrease in lymphocyte cAMP content and appeared to be neither an inhibitor nor a substrate for S-adenosylhomocysteine hydrolase. Consistent with this latter result, l-homocysteine thiolactone had no effect on the inhibition of LMC by c 9Ado. Neither the inhibition of LMC by c 9Ado nor the metabolic formation of c 9ATP in lymphocytes was affected by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), indicating that c 9Ado is not a substrate for adenosine deaminase. 5-Iodotubercidin, a non-competitive inhibitor ( K 15 = 9 nM, K ii = 20 nM) of adenosine kinase, prevented the above effects of c 9Ado on lymphocyte function, c 9ATP formation, and ATP levels. Either complete preservation (with coformycin) or partial replenishment (with adenosine plus EHNA) of ATP levels in c 9Ado-treated lymphocytes resulted in partial restoration of cytolytic function to cells containing large amounts of c 9ATP. These results suggest that c 9Ado is inhibitory to LMC both because it causes a decrease in the absolute concentration of ATP within the cytolytic lymphocytes and because it permits the establishment within these cells of an unfavorable c 9ATP: ATP ratio which impedes the utilization of ATP in a reaction essential to the execution of this lymphocyte function.

Characterization of the ( [formula omitted] in a membranous preparation from the optic ganglion of the squid ( Loligo pealei)

(1) A membrane fraction enriched in (Na + + K +)-ATPase (EC 3.6.1.3) was obtained from optic ganglia of the squid ( Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an (Na + + K +)-ATPase specific activity of approx. 2 units/mg and was >95% ouabain-sensitive. (2) The (Na + + K +)-ATPase had a K m for ATP of 0.42 ± 0.04 mM and a pH optimum of 7.0. It was inhibited by ouabain with a K i of 0.32 ± 0.04 μ M . (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with NaCl + KCl = 300 mM . (4) The Mg 2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the K m for Mg 2+ was 0.86 ± 0.10 mM , and at 6 mM ATP, the K m was 1.86 ± 0.44 mM . High levels of Mg 2+ caused inhibition of hydrolysis. (5) The interactions of Na + and K + were examined over a range of conditions. K + levels caused modulations in the Na + dependence in the range of 1–150 mM. (6) The (Na + + K +)-ATPase prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.

Secondary bioenergetic hypoxia. Inhibition of sulfation and glucuronidation reactions in isolated hepatocytes at low O2 concentration.

The O2 dependence of cytochrome oxidase, cellular ATP/ADP ratio, and drug sulfation and glucuronidation were studied in isolated hepatocytes to examine secondary biochemical changes of hypoxia related to decreased cytochrome oxidase function. A decrease in cytochrome oxidase function as well as in cellular ATP/ADP ratio occurred at low O2 concentrations and their O2 dependencies gave p50 values of 3.2 and 2.8 microM O2, respectively. Changes in the ATP/ADP ratio corresponded directly with changes in the oxidation-reduction state of cytochrome oxidase. The p50 value for sulfation of acetaminophen (2.5 microM O2) corresponded with the values for ATP/ADP ratio and cytochrome oxidase. Addition of compounds that lowered cellular ATP/ADP ratio caused a corresponding inhibition of sulfation. Selective use of ethionine, an adenosyl-trapping agent, demonstrated that the O2 dependence of sulfation related directly to a decrease in the absolute ATP concentration. The p50 value for glucuronidation (5.2 microM O2) was higher than that for sulfation, but in spite of this difference, glucuronidation also correlated well with cellular ATP/ADP ratio when this ratio was decreased by either O2 limitation or metabolic inhibitors. These results demonstrate that the predominant limitation of drug sulfation and glucuronidation during hypoxia is due to decreased cytochrome oxidase function.