An ATP pool associated with adenyl cyclase of brain tissue

R adioactive adenine has been frequently used to label cyclic adenosine 3', 5'-monophosphate (cAMP) in diverse cells and tissues (K uo and D e R enzo , 1969; S himizu , D aly and C reveling , 1969, B rooker , 1971). We have previously reported that cAMP from the guinea-pig cerebral cortex is derived from a precursor pool of adenine nucleotides which is more highly labelled than the bulk cellular ATP (S himizu , C reveling and D aly , 1970a). The present study was carried out to throw additional light on the property of the precursor pool. Levels of cAMP and ATP and specific activities of [¹⁴C]cAMP and [¹⁴C]ATP were determined simultaneously in cerebral slices which were prelabelled with [¹⁴C]adenine and subsequently incubated with veratridine, histamine or adenosine.     

Increased ATP and creatine phosphate turnover in phagocytosing mouse peritoneal macrophages.

Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Metabolism of adenine nucleotides in thymocyte cytoplasm and subcellular fractions after treatment with concanavalin A, papaverine and adenosine

Studies with rat thymocytes labeled with [14C]adenine and fractionated by digitonin treatment revealed that the cytoplasm of these cells contains about 60% of the total adenine nucleotide pool with a higher ATP/ADP ratio and metabolic activity as compared with the structural components. The incorporation of [14C]adenine and [14C]adenosine into thymocyte adenine nucleotides results in predominant labeling of cytoplasmic ATP, in which the specific radioactivity of this nucleoside triphosphate is two and three times as high as in subcellular structures. Concanavalin A decreases the ATP level in thymocytes without changing its specific radioactivity. This compound does not influence the total content and amount labeled adenine nucleotides in the structural fraction. Papaverine accelerates the catabolism of ATP, mainly in thymocyte cytoplasm and, in a lesser degree, in its structural fraction. In each fraction the papaverine-induced catabolism of ATP is localized in the compartment which is more intensively labeled with [14C]adenine than the whole fractionation ATP pool. Adenosine markedly accelerates adenine nucleotide catabolism in the cytoplasmic and structural fractions of thymocytes; however, only in the first one of them this acceleration is due to ATP elevation. Papaverine and adenosine do not directly influence either the content or specific radioactivity of adenine nucleotides of the structural fraction isolated from [14C]adenine-labeled thymocytes.     

Characteristics of adenosine activity on adenine nucleotide metabolism of rat thymocytes

The effects of adenosine on adenine nucleotide metabolism in [14C]adenine-labeled rat thymocytes were studied. It was shown that adenosine increases the intracellular pool of adenine nucleotides, predominantly ATP, which is accompanied by marked acceleration of their catabolism and a release of labeled products (especially inosine, hypoxanthine and adenosine) from the thymocytes. The effect of adenosine depends on its concentration and manifests itself already at 10(-6) M. 2-Deoxycoformycin partly relieves the effect of adenosine on adenine nucleotide metabolism. Exogenous deoxyadenosine, inosine, hypoxanthine and adenine, unlike adenosine, do not significantly affect the adenine nucleotide catabolism and the label release from the cells. All the effectors under study strongly increase inosine transport from the thymocytes, and inhibit, with the exception of adenosine, the hypoxanthine release from the cells.     

Role of the intramitochondrial adenine nucleotides as intermediates in the uncoupler-induced hydrolysis of extramitochondrial ATP.

1. A formula is given that describes the appearance of [14C]ATPADP outside the mitochondria after the addition of [14C] 1atp during the steady-state uncoupler-induced hydrolysis of extramitochondrial ATP. If the transported adenine nucleotides equilibrate with the intramitochondrial pool, [14C]ADP0 would be expected to appear with a lag phase that corresponds with the time needed for the radioactive labelling of the intramitochondrial adenine nucleotide pool. 2. The rates of formation of [14C]ADP outside the mitochondria after addition of [14C]ATP during the steady-state uncoupler-induced ATP hydrolysis catalysed by rat-liver mitochondria at 0 degree C were measured. 3. In the presence of carbonyl cyanide m-chlorophenylhydrazone the time course of the [14]ADPo formation was the same as that predicted on the basis of the above assumption. 4. In the presence of the less effective uncoupler, 2,4-dinitrophenol, the time course of [14C]ADPo formation was not consistent with the theoretical predictions: no lag phase was present and the measured rate was higher than the maximal calculated rate. These results can be explained by assuming a functional interaction between the adenine nucleotide translocator and the mitochondrial ATPase (F1). 5. It is concluded that under phosphorylating as well as dephosphorylating conditions, the adenine nucleotide translocator and the mitochondrial ATPase can be functionally linked to catalyse phosphorylation or dephosphorylation of extramitochondrial ADP or ATP, without participation of the intramitochondrial adenine nucleotides.     

An ATP pool with rapid turnover within the cell membrane

Seven-day-old cultures of rat leg muscle cells were double labelled by addition of [¹⁴C] adenine in the culture medium (2 hrs 15 mins) and followed by addition of [³²P] phosphate (15 min). The specific activity (S.A.) of the isolated cyclic [¹⁴C] adenine 3′ – 5′ monophosphate (cAMP) was similar to that of the bulk ATP. The S.A. of [³²P] from cAMP was, however, higher than that of bulk ATP. The S.A. of [³²P] from cAMP could be further modified by prevention of normal muscle cell fusion. It is probable that the cAMP with high [³²P] S.A. was synthesized from a cell membrane pool of ATP with rapid turnover.     

Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.     

A new chromatographic method using immobilized acriflavin for measuring cyclic AMP in cells prelabeled with radioactive adenine.

A method using the principle of charge-transfer chromatography has been developed for the determination of cyclic AMP levels in intact prelabeled cells. The ATP pool was prelabeled by incubating the cells in the presence of radioactive adenine. The cyclic AMP formed from ATP was extracted with HC10(4) and separated from adenine and other adenosine-related nucleotides by chromatography on acriflavin-Sephadex G-25. This method provides a rapid and sensitive isolation of cyclic AMP with high recovery (95-100%) and low blnks. Further, no contamination of the cyclic AMP fractions was found by either adenine or adenosine nucleotides such as ATP, ADP or AMP. This procedure is applicable to a variety of cell or tissue systems.     

Effect of papaverine on the absorption and metabolism of 14C- adenosine and 14C-adenine in thymocytes

Some peculiarities of adenosine and adenine nucleotide metabolism in rat thymocytes were investigated. It was shown that the uptake of labelled adenosine or adenine by thymocytes is markedly inhibited by papaverine due to the decrease of the adenylate kinase activity, on the one hand, and to the acceleration of ATP catabolism and inosine and hypoxanthine release into the environment, on the other. ATP catabolism occurs in a special compartment which in [14C] adenosine and [14C] adenine prelabelled thymocytes has a higher specific radioactivity as compared with the whole cell. In [14C] adenine-prelabelled thymocytes and extracellular medium, papaverine does not influence the content but increases the specific radioactivity of adenosine.     

P1-(5'-adenosyl)-P2-N-(2-mercaptoethyl)diphosphoramidate. An affinity reagent for demonstrating the presence of Tyr-beta 311 at the hydrolytic site of F1-ATPase

The compound P1-(5'-adenosyl)-P2-N-(2-mercaptoethyl)diphosphoramidate (AMEDA) was synthesized as an ATP analogue for in situ reaction with the 4-nitro-2,1,3-[14C]benzoxadiazolyl group (NBD) in the labeled F1-ATPase (F1). AMEDA was found to reactivate O-[14C]NBD-F1 via a dual-path mechanism. The principal path involves the binding of AMEDA at a site in F1 with Kd = 14.5 microM and subsequent reaction with the [14C]NBD label. The second slower path involves the direct biomolecular reaction of AMEDA with the radioactive label on F1. The rate of reactivation of O-[14C]NBD-F1 by AMEDA was decreased by ADP or ATP which competes with the ATP analogue for binding to the labeled enzyme. The reaction product was found to contain one adenine group, two phosphate groups, and one [14C]NBD label per molecule as expected from the structure of the compound AMEDA-[14C]NBD. Purified AMEDA-[14C]NBD was found to bind to unlabeled F1 with Kd = 2 microM. These observations demonstrate the in situ reaction of bound AMEDA with the nearby [14C]NBD label attached to Tyr-beta 311 and support the assumed presence of Tyr-beta 311 near the phosphate groups of ATP bound at the hydrolytic site of F1-ATPase. The possible locations of Tyr-beta 364, His-beta 427, and Tyr-beta 345 relative to Tyr-beta 311 in F1 are discussed, and the validity of the previously proposed model for F1-ATPase with one hydrolytic site assisted by two auxiliary sites is examined and compared with that of the widely accepted alternating sites model.     

Fatty acid uptake and catecholamine-stimulated phospholipid metabolism in immortalized airway epithelial cells established from primary cultures

The incorporation of [14C]oleic and [14C]linoleic acid into phospholipids and neutral lipids was compared in two recently immortalized airway epithelial cell lines. In addition, the effects of adrenergic stimulation on phospholipid turnover was examined. Both cell lines readily incorporated the fatty acids into all phospholipid and neutral lipid fractions. Isoproterenol (1 microM) induced Ca2+ transients in both cell lines, indicating a functional beta-adrenergic response. Epinephrine (10 microM; 15 min) stimulation of cells prelabeled with [14C]linoleic acid increased the percentage of label in phosphatidylcholine in one cell line. Lipid metabolism can now be extensively studied in human airway epithelia.     

1-Methyladenine production from ATP by starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance in starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves. We have shown previously that GSS causes a reduction in the intracellular levels of ATP coincident with 1-MeAde production. The present study examined whether the adenine molecule of 1-MeAde is directly derived from ATP. When isolated follicle cells from the starfish Asterina pectinifera were preloaded with [U-14C]adenine or [U-14C]adenosine, there was an increase in the intracellular levels of radiolabeled adenine nucleotides, particularly ATP. Following further incubation with GSS, the intracellular levels of radiolabeled ATP decreased, concomitant with a marked increase in the levels of [14C]1-MeAde in the medium. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeAde produced. However, there was no change in the levels of ADP and AMP regardless of the presence or absence of GSS. These findings strongly suggest that 1-MeAde is synthesized from ATP as a substrate in follicle cells under the influence of GSS. Furthermore, using [methyl-3H]methionine, the methyl group of 1-MeAde was found to be derived from methionine. Thus GSS appears to stimulate the synthesis of 1-MeAde from ATP via the methylation process in starfish ovarian follicle cells.     

A note on the synthesis of 5'-AMP ester of tris (hydroxymethyl)aminomethane by rat liver plasma membrane.

The alcohol-AMP synthesizine enzyme of rat liver plasma membrane also synthesizes the 5'-AMP ester of tris(hydroxymethyl)aminomethane as judged by the use of [alpha-32P] ATP and [U-14C] ATP. This synthetic process may decrease significantly the concentration of ATP during incubation.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures

The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84-88% of the labeled adenine nucleotides, ADP 11-13%, and AMP 1-3%. The turnover of 32P-adenine nucleotides and 3H-nucleotides was biphasic and virtually identical; there was an initial fast phase with a t1/2 of 3.5-4.5 h and a slow phase with a half-life varying from 7 to 17 days, depending upon the particular cell preparation. The t1/2 of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half-life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half-lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P-labeled compounds and relatively three times as much catecholamine as 3H-labeled compounds.     

Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2-3H]-adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5'-monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5'-triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5'-monophosphate [AMP] deaminase and adenosine deaminase) and of 5'-amino-5'-deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine-induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5'-triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.     

Adenine metabolism of Ehrlich mouse ascites cells in proliferating and resting phase of tumor growth.

The incorporation of 14C from [U-14C]adenine into the pools of purine nucleotides, nucleosides and bases in Ehrlich mouse ascites cells (EMAC1) during the proliferating and resting phases of tumor growth was compared. In the proliferating phase the total 14C incorporation into purine pools is much faster than in the resting phase. The ATP turnover as well as the purine breakdown to hypoxanthine and uric acid are increased in the proliferating phase. That corresponds to previous findings on higher nucleotide pool sizes and higher ATP yield and ATP-consuming processes in this growth period.