Intracellular Adenine Nucleotide Research Articles

THE PROTECTIVE ACTION OF INOSINE ON ISOLATED ARTERIES IN HYPOXIA

1 The pressor responses to injected noradrenaline (NA) of isolated perfused femoral or renal arteries of the rabbit were studied.2 Vascular smooth muscle is relatively resistant to hypoxia. A combination of hypoxia and dinitrophenol (DNP) respiratory uncoupling was necessary to abolish the pressor response to NA. Loss of the pressor response was assumed to result from decreased capacity of arteries to form adenosine 5'-triphosphate (ATP). Reperfusion of the hypoxic arteries with oxygenated medium resulted in recovery of the pressor response to NA.3 Inclusion of inosine (10 mM) in the hypoxic perfusion medium increased significantly the rate and extent of post-hypoxic recovery of the pressor response to NA.4 Whereas the presence of inosine in the hypoxic perfusion medium aided post-hypoxic recovery, inosine had no direct action on the pressor dose response to NA. Therefore, the action of inosine was protective as opposed to direct.5 The protective action of inosine did not involve potentiation of NA binding to NA-adrenoceptor sites (the equilibrium coefficient, K(eq) for NA-receptor interaction was unaltered by hypoxia and/or inosine).6 The results are discussed in terms of a presumptive mechanism whereby inosine is believed to act by maintaining intracellular adenine nucleotide concentrations in hypoxia.

Action of purine nucleosides on the release of intracellular enzymes from rat lymphocytes

Rat lymphocytes incubated under hypoxic conditions in vitro show a time-dependent release of intracellular enzymes. As reported previously, enzyme release (lactate dehydrogenase) is decreased by metabolites, notably ATP and glucose, that contribute towards lymphocyte energy metabolism. The action of purine nucleosides in relation to enzyme release was investigated. Inosine was shown to decrease significantly lactate dehydrogenase release, whereas adenosine exerted a supportive action only at concentrations less than 0.5 mmol/l. Inosine decreased enzyme efflux maximally at concentrations of at least 2–3 mmol/l. The mechanism of inosine action was deduced to be primarily ribose 5-phosphate formation and its subsequent metabolism by energy-yielding pathways. Inosine was presumed also to enter the purine ‘salvage pathway’ and thereby maintain intracellular adenine nucleotide pools.

Effect of physiological and pharmacological doses of insulin on the intracellular glucose, glycogen, glucose transport and adenine nucleotide levels in isolated hepatocytes

Effect of physiological and pharmacological doses of insulin on the intracellular glucose, glycogen, glucose transport and adenine nucleotide levels in isolated hepatocytes

Increased incorporation of adenosine into adenine nucleotide pools in serum-deprived mammalian cells.

The effects of serum deprivation on the incorporation of adenosine into the intracellular adenine nucleotide pools by several mamalian cell lines were studied. Cells arrested in the G1 phase of the cell cycle showed increased incorporation of exogenous adenosine into their adenine nucleotide pools as compared with growing cells. This phenomenon is unexpected because salvage pathways from all other preformed nucleosides and bases as well as the de novo synthesis of adenine nucleotides is decreased after arrest of growth by serum deprivation. The incorporation of adenosine into adenine nucleotides may serve as an intracellular signal in the regulation of growth in mammalian cells.

Inhibition of hepatic lipogenesis by adenine nucleotides

Incubation of liver slices and isolated liver cells with adenosine cyclic-3',5'-monophosphate at concentrations which inhibit lipogenesis was found to expand the pool size of the noncyclic adenine nucleotides in the intact cells of the preparations. This observation led to studies which demonstrated that adenosine and adenosine-5'-monophosphate also inhibited lipogenesis and expanded the adenine nucleotide pool size. It is proposed but not proven that the increase in intracellular nucleotides produced by adenosine-5'-monophosphate, adenosine cyclic-3',5'-monophosphate, and adenosine may have an adverse effect upon the synthesis of fatty acids. Because of the expansion of the adenine nucleotide pool size, high concentrations of adenosine cyclic-3',5'-monophosphate should not be used to investigate the mechanism responsible for hormonal regulation of lipogenesis. As an added complication, exogenous adenosine-5'-monophosphate was found to produce a small but significant increase in the intracellular concentration of adenosine cyclic-3',5'-monophosphate of isolated liver cells. This effect also may be a factor in the inhibition of lipogenesis by adenosine-5'-monophosphate. Low concentrations of N6, O2'-dibutyryl adenosine cyclic-3',5'-monophosphate were found to inhibit lipogenesis without increasing the intracellular adenine nucleotide content of either liver slices or isolated liver cells. It is concluded that studies on the mechanism of glucagon regulation of lipogenesis should be carried out with glucagon or low concentrations of N6, O2'-dibutyryl adenosine cyclic-3',5'-monophosphate.

Relation of growth and protein synthesis to the adenylate energy charge in an adenine-requiring mutant of Escherichia coli.

When Escherichia coli K-12 strain PC 0294, which is unable to synthesize adenine nucleotides, thereonine, proline, and leucine, was starved for adenine, the concentrations of ATP, ADP, and AMP fell rapidly. The adenylate energy charge was not affected until the adenine nucleotide pool fell to about 30% of its normal value. Similarly, cells in an adenine-limited chemostat grew at intracellular adenine nucleotide pool values as low as 30% of normal, but the energy charge in all cases was approximately 0.90. Incorporation of [14C]leucine into protein in adenine-starved cells continued rapidly as long as the energy charge was in or near its normal range, even when the concentration of ATP was 30% to 10% of its normal value. When glucose was added to cells that had been resuspended in medium lacking both glucose and adenine, the energy charge rose rapidly but the concentration of ATP fell, presumably because of nucleic acid synthesis. The rate of [14C]leucine incorporation into protein rose rapidly while adenine nucleotide concentrations fell, and then declined roughly in parallel with the energy charge. At a given value of energy and of total concentration of adenine nucleotides, the rate of protein synthesis may, of course, vary with the concentrations of precursors and modifiers; thus, the rate cannot be predicted from knowledge only of the energy charge. Our results suggest, however, that the rate of protein synthesis and the capacity for growth are much more sensitive to changes in the value of the energy charge than to changes in the concentration of ATP. Growth occurs when the ATP concentration is reduced to one-third of its normal value, but has not been observed when the energy charge has fallen by as much as 10%.

Stimulated Formation of Cyclic Adenosine 3′:5′-Monophosphate by Aspartate and Glutamate in Cerebral Cortical Slices of Guinea Pig

Abstract The content of cyclic adenosine 3':5'-monophosphate (cyclic AMP) in guinea pig cerebral cortical slices increases approximately 40-fold during a 10-min incubation and 80-fold during a 20-min incubation in a medium containing 10mm l-glutamate. A similar increase was observed in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides, labeled by a prior incubation with radioactive adenine. The stimulatory effect of l-glutamate on formation of radioactive cyclic AMP was found with a concentration as low as 0.05 mm, and concentrations of l-glutamate required for the half-maximal and maximal effects were about 1.5 and 10 mm, respectively. At 10 mm concentration, l-aspartate was as good as, and d-aspartate, d-glutamate, dl-α-aminoadipate, and l-alanine were weaker than, l-glutamate as a stimulant for the cyclic AMP formation. All other amino acids and metabolites of glutamic acid tested were virtually ineffective. Mutual potentiation of effects was observed when aspartate was added together with either adenosine or histamine, but not with glutamate, veratridine, or ouabain. The effect of the two amino acids were completely blocked by theophylline, but not by omission of Ca2+ or by addition of cocain, the conditions known to inhibit the depolarization-elicited formation of cyclic AMP. The formation of radioactive cyclic AMP stimulated by l-glutamate was allowed to return to a half of the stimulated levels within 20 min after removal of the glutamate by washing, and then was restimulated either by adenosine, l-aspartate, or l-glutamate. These properties of the amino acids suggest that the amino acids may be a new class of stimulants which is different from biogenic amines, adenosine, and depolarizing agents as stimulants for cyclic AMP formation in incubated brain slices.

Adenosine metabolism in Micrococcus sodonensis.

Enzymes required for the synthesis of purine ribonucleotides from exogenous adenosine, inosine, and guanosine were shown to be present constitutively in Micrococcus sodonensis. An indirect pathway for the synthesis of adenine nucleotides from adenosine was suggested by the high intracellular adenosine deaminase activity. Concentrations of intracellular adenine nucleotides were elevated in the presence of exogenous adenosine and under those conditions direct measurements of the activity of the pathway for de novo purine ribonucleotide biosynthesis indicated that the activity of that pathway was almost completely inhibited.

Cyclic Adenosine 3′,5′-Monophosphate in Guinea Pig Cerebral Cortical Slices: I. FORMATION OF CYCLIC ADENOSINE 3′,5′-MONOPHOSPHATE FROM ENDOGENOUS ADENOSINE TRIPHOSPHATE AND FROM RADIOACTIVE ADENOSINE TRIPHOSPHATE FORMED DURING A PRIOR INCUBATION WITH RADIOACTIVE ADENINE

Abstract Cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulates in guinea pig cerebral cortical slices during incubations with histamine, histamine-norepinephrine, adenosine, adenosine-histamine, adenosine-norepinephrine, adenosine-histamine-norepinephrine, veratridine, and veratridine-histamine. The rate of increase of cyclic AMP levels in response to various agents appears to be limited to some extent by the rate of diffusion of small molecules into the extracellular space of the slices. The diffusion of [3H]sorbitol occurred with a t½ of l3 min. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides, labeled by a prior incubation with radioactive adenine, occurred on similar time courses and to similar extents. Radioactive cyclic AMP represents a small but relatively constant fraction (7 to 13%) of the total amount of cyclic AMP. During stimulation with histamine, however, the initial accumulation of radioactive cyclic AMP represents a significantly smaller fraction of the total amount of cyclic AMP. Except in this instance, the data provide no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that can serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment or compartments appear to be uniformly labeled by incubation with radioactive adenine and to represent about 30% of the total adenine nucleotides of the slice. Conversion of radioactive adenine to cyclic AMP is proportional to the steady state levels of the cyclic nucleotide and occurs at rates of up to 10 pmoles per mg of protein per min in the continued presence of stimulatory agents.

Accumulation of adenosine 3':5'-cyclic monophosphate in clonal glial cells: labeling of intracellular adenine nucleotides with radioactive adenine.

The accumulation of endogenous adenosine 3':5'-cyclic phosphate (cAMP) and of [(14)C]cAMP, derived from nucleotides labeled by prior incubation of cells with [(14)C]adenine, has been investigated in four glial cell lines from rats. The following results are similar to data reported for brain slices: (i) rat glial cells contain a system that readily incorporates [(14)C]adenine into nucleotides that serve as precursors for cAMP. (ii) norepinephrine stimulates accumulation of both [(14)C]cAMP and endogenous cAMP, and (iii) the phosphodiesterase inhibitors, papaverine and isobutylmethylxanthine, are effective in enhancing the stimulatory effect of norepinephrine. In contrast to results reported for brain slices, histamine, veratridine, and adenosine, the purine either alone or with a biogenic amine, do not cause an enhanced accumulation of cAMP in rat glial cells, and repetitive accumulations of cAMP can be elicited during a series of restimulations of the cells with norepinephrine. The magnitude of the accumulation of cAMP elicited by the catecholamine decreases markedly by the fifth restimulation in the absence of, but only slightly in the presence of, papaverine. In glial cells, a large portion of the [(14)C]adenine is incorporated into intracellular compounds that do not serve as precursors of [(14)C]cAMP.

Nucleotide metabolism during differentiation in Dictyostelium discoideum.

Energy metabolism in Dictyostelium discoideum was studied by following the incorporation of [(3)H]adenine and [(32)P]Pi into the intracellular adenine nucleotides at two stages of differentiation. That the levels of nucleotides measured represented levels present in vivo was shown by demonstrating that the concentrations determined from cells quickly frozen in liquid nitrogen were identical to those from cells harvested and subsequently killed in perchloric acid. In addition, significant compartmentalization among the nucleoside triphosphates was not observed after 30 min, since uridine triphosphate, guanosine triphosphate, and adenosine triphosphate (ATP) were at radioactive equilibrium after exposure to [(32)P]Pi. At the sorocarp stage of differentiation, the dominant role of adenylate kinase activity is indicated by the observations that (i) adenylate kinase activity was found to be present, (ii) the specific radioactivities of the beta- and gamma-phosphates of ATP were equal, (iii) the increase in specific radioactivity of adenosine diphosphate was one-half that of ATP in the presence of [(32)P]Pi, and (iv) the steady-state equilibrium constant calculated (0.69) from the nucleotide levels equaled that of the apparent equilibrium constant reported in the literature (0.70). By using the same criteria, adenylate kinase activity was not the predominant reaction establishing the adenine nucleotide levels in cells at the pseudoplasmodium stage of differentiation.

Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell.

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90 degrees light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox --> condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90 degrees light-scattering increase, which also occurs at this time, showed a t (1/2) of only 25 sec which agreed temporally with a slower orthodox --> maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox --> condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.

The effects of ethionine treatment on the metabolism of liver mitochondria

The administration of the methionine analog, ethionine, to female rats lowers the mitochondrial as well as the total intracellular level of ATP in the liver. The mitochondrial ADP/O, state 4 Q 02, i.e., the respiratory rate in the absence of ADP, Ca 2+- and water-activated ATPase remain unchanged up to 4 hr after ethionine treatment. However, the respiratory control index, state 3 Q 02, i.e., the respiratory rate in the presence of ADP, and DNP-activated ATPase are considerably lower than the control values. The preincubation of the mitochondria in a medium favorable to K + transport will partially restore the activity of the DNP-activated ATPase. DNP-stimulated respiration is normal when potassium succinate is the substrate but is inhibited with potassium glutamate as substrate. Ethionine doses of 0.25 mg/g body wt and 0.5 mg/g body wt have little effect on the respiratory control index or state 3 Q 02 of rat liver mitochondria, although the latter dose reduces the hepatic ATP level to approximately 35% of the control value; doses of 0.75 mg/g body wt and 1 mg/g body wt reduce the ATP level to about 20% of the control value and produce inhibition of the respiratory control index and state 3 Q 02. Therefore, it seems that mitochondria can function quite normally in respect to the control and generation of energy via respiration in the presence of a large decrease in intracellular adenine nucleotides. However, when the ATP concentration falls below 35% of the control value, changes in mitochondrial metabolism become apparent and the data suggest that these changes may be due to inhibition of the transphosphorylation reactions.

Biochemical energetics of simulated platelet plug formation. Effect of thrombin, adenosine diphosphate, and epinephrine on intra- and extracellular adenine nucleotide kinetics.

Washed human platelets were incubated in a modified Ringer's solution, pH 7.1, at 37 degrees C for 1 hr. Intracellular basal levels for glycogen, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and orthophosphate were 31.1, 2.52, 1.39, 0.36, and 1.2 mumoles/ml of platelets, respectively. Extracellular ATP, ADP, and AMP remained fairly constant and represented 4, 2, and 4% of total adenine nucleotide content. Total adenine nucleotide content remained unchanged during the period of control incubation. Glycogen depletion was 17.8 mumoles/ml at the end of 1 hr; lactate production was 20.7 mumoles/ml per hr. In the presence of glucose, lactate production increased 100%, and glycogen depletion was spared 13%. Approximately 55% of glucose or glycogen fuel was converted to lactate. The agglutinating agents, thrombin, ADP, and epinephrine, resulted in increased glycogen depletion and lactate production both in the presence and absence of glucose. The effect of thrombin was greater than epinephrine. The effect of epinephrine was greater than ADP. All three agglutinating agents resulted in loss of high energy phosphates (net decline in adenine nucleotides) with release of adenine nucleotides into the extracellular environment. The effect of thrombin was greater than ADP. The effect of ADP was greater than epinephrine. In experiments with ADP addition, significant quantities of ADP were converted to AMP extracellularly. In experiments with thrombin and epinephrine appreciable quantities of extracellular orthophosphate were taken up by plateletes and could not be accounted for by changes in intracellular orthophosphate or adenine nucleotide. Sufficient ADP was released during exposure to thrombin and epinephrine to account for platelet agglutination. Changes in intracellular adenine nucleotides and orthophosphate could be correlated with the activation of regulator glycogenolytic and glycolytic enzymes.

Studies of Baker's yeast metabolism: II. The role of adenine nucleotides and inorganic phosphate in the control of respiration during alcohol oxidation

The respiratory inhibition following within 40 seconds after the addition of ethanol to a starved baker's yeast cell suspension has been studied by the analysis of intracellular adenine nucleotides and phosphate. Experiments with chemicals like glucose, 2-deoxyglucose, or glucose in the presence of iodoacetic acid have revealed that the observed respiratory inhibition is caused by the unavailability of adenosine 5′-diphosphate. By the use of a suitable phosphate trap, such as 2-deoxyglucose or glucose, it has been possible to demonstrate in the alcohol-oxidizing yeast a respiratory inhibition mediated by the lack of inorganic phosphate. A true Crabtree effect brought forth by glucose could not be demonstrated, although a net inhibition of respiratory rate has been shown by the use of 2-deoxyglucose or by glucose in the presence of iodoacetic acid. The data have been discussed in relation to the general problem of control of respiratory metabolism in intact cell systems.