The metabolism of the erythrocyte. XVIII. Inhibition of nucleotide synthesis in human erythrocytes by adenosine

1968 ◽  
Vol 46 (5) ◽  
pp. 445-450 ◽  
Author(s):  
S. V. Manohar ◽  
M. H. Lerner ◽  
D. Rubinstein
Keyword(s):  
Adenosine Deaminase ◽  
Small Proportion ◽  
Inhibitory Effect ◽  
Human Erythrocytes ◽  
Intracellular Concentration ◽  
Intracellular Level ◽  
Nucleotide Synthesis ◽  
Adenylic Acid

Incubation of human erythrocytes with glucose and 1.5 mM adenine for 12 h doubles the intracellular level of ATP because of synthesis of the nucleotide from the purine. The effect of the nucleosides inosine, guanosine, and adenosine on the synthesis of nucleotide labile phosphate during the incubation was studied. Inosine and guanosine had no effect, but adenosine, in concentrations between 10 and 20 mM, inhibited the increase in labile phosphate and the incorporation of adenine-8-14C into ATP. The adenosine has no effect on the slight incorporation of adenine-8-14C into, or the intracellular concentration of, ADP or AMP. The inhibition is not due to the formation of ammonia and (or) inosine resulting from the deamination of adenosine, nor is it due to the change of pH produced by the ammonia. Adenosine must be present to produce its inhibitory effect and the inhibition can be reversed by removal of the nucleoside, either by washing the cells or through the action of the erythrocyte adenosine deaminase. It thus appears that adenosine produces its effect when it is present in concentrations too great to be completely deaminated. A small proportion (0.5%) of the added adenosine is incorporated into the cellular nucleotides, more than half into IMP, resulting in a marked increase in the concentration of this nucleotide. It is suggested that adenosine acts by inhibition of the synthesis of either 5-phosphoribosyl-1-pyrophosphate or adenylic acid.

scholarly journals Intracellular Mg2+regulates ADP phosphorylation and adenine nucleotide synthesis in human erythrocytes

1998 ◽  
Vol 274 (5) ◽  
pp. E920-E927 ◽  
Author(s):  
Sarah Page ◽  
Michael Salem ◽  
Maren R. Laughlin
Keyword(s):  
Adenine Nucleotide ◽  
Phosphoglycerate Kinase ◽  
Pentose Phosphate ◽  
Human Erythrocytes ◽  
Maximal Velocity ◽  
Nucleotide Synthesis ◽  
Ion Concentrations ◽  
Net Flux ◽  
Normal Fluctuations ◽  
Phosphorylation Rate

13C- and31P-NMR were used in methylene blue-treated human erythrocytes to determine the dependence on intracellular Mg2+concentration ([Mg2+]i) of the pentose phosphate pathway (PPP), the glycolytic pathway, and adenine nucleotide synthesis. The PPP flux had an [Mg2+]iat half-maximal velocity ([Mg2+]i,0.5) of 0.02 mM, well below the physiological range (0.2–0.7 mM). Flux through the PPP was reduced at higher [Mg2+]ias flux through phosphofructokinase was increased ([Mg2+]i,0.5= 0.16 mM). [Mg2+]i,0.5of phosphoglycerate kinase flux, which equals net ADP phosphorylation rate, was 0.27 mM, well within the physiological [Mg2+]irange. The rate of adenine nucleotide synthesis from [2-13C]glucose-derived ribose 5-phosphate and exogenous adenine also exhibited dependence on [Mg2+]ibut was not saturable up to 1.6 mM. Therefore, net flux through the PPP and glycolytic pathways in erythrocytes is not strongly dependent on [Mg2+]iat physiological ion concentrations, but both ADP phosphorylation and adenine nucleotide synthesis are likely to be regulated by normal fluctuations in [Mg2+]i.

Quantitative measurement of adenosine deaminase from human erythrocytes

1975 ◽  
Vol 63 (3) ◽  
pp. 323-333 ◽  
Author(s):  
W. Korber ◽  
Eva B. Meisterernst ◽  
G. Hermann
Keyword(s):  
Adenosine Deaminase ◽  
Quantitative Measurement ◽  
Human Erythrocytes

The mobility of intramembrane particles in non-haemolysed human erythrocytes. Factors affecting acridine-orange-induced particle aggregation

1986 ◽  
Vol 86 (1) ◽  
pp. 57-67
Author(s):  
G. Lelkes ◽  
I. Fodor ◽  
G. Lelkes ◽  
S.R. Hollan
Keyword(s):  
Acridine Orange ◽  
Intracellular Ph ◽  
Lateral Diffusion ◽  
Inhibitory Effect ◽  
Human Erythrocytes ◽  
Particle Aggregation ◽  
Experimental Conditions ◽  
Factors Affecting ◽  
Cross Links ◽  
Phosphate Buffered Saline

It has previously been shown that reversible intramembrane particle aggregation can be induced in non-haemolysed human erythrocytes. This phenomenon, which can be induced by the cationic dye Acridine Orange, has been further investigated using different experimental conditions that are expected to influence the rate of aggregation of the particles. In addition to the concentration of the dye, the rate of aggregation was also found to be dependent on the extracellular and intracellular pH, as well as on the type of buffer used. While lowering the pH of the Acridine Orange solutions resulted in decreased particle clustering, low intracellular pH increased and elevated intracellular pH decreased particle aggregation. Furthermore, at a given dye concentration and a given pH, Acridine Orange caused more intense aggregation in Tris-buffered saline than in isotonic phosphate buffer or phosphate-buffered saline. Under appropriate conditions Acridine Orange caused significant particle aggregation at concentrations as low as 0.25 mM within 30 s. During this period only discocyte-stomatocyte transformation occurred; neither agglutination nor vesiculation of the erythrocytes could be detected. Treatment of the erythrocytes with Diamide (Serva), which cross-links spectrin via disulphide bridges and thereby reduces lateral diffusion of integral membrane proteins over large distances, had no inhibitory effect on Acridine-Orange-induced particle aggregation. Heating the erythrocytes to 50 degrees C, at which temperature denaturation of spectrin and fragmentation of the erythrocytes occur, and subsequently incubating them in Acridine Orange at room temperature, caused an almost maximal rate of particle aggregation within 10–30 s, without haemolysis. The possible mechanism and significance of the particle aggregation phenomenon are discussed.

Effects of adenosine 5′-monophosphate and adenosine 3′,5′-cyclic monophosphate on l cells

1975 ◽  
Vol 19 (2) ◽  
pp. 305-313
Author(s):  
J. Taylor-Papadimitriou ◽  
T. Karemfyllis ◽  
A. Eukarpidou ◽  
G. Karamanlidou
Keyword(s):  
Cyclic Amp ◽  
Adenine Nucleotides ◽  
Inhibitory Effect ◽  
S Phase ◽  
Breakdown Product ◽  
Intracellular Level ◽  
Effective Inhibitor ◽  
Growth Inhibitory Effect ◽  
L Cells ◽  
Growth Inhibitory

The adenine nucleotides, 5′-AMP and 3′,5′-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5′-AMP, and rates of uridine transport are increased after incubation with either 5′-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.

scholarly journals The Elevation of Adenosine-Triphosphate Levels in Human Erythrocytes

Blood ◽  
1973 ◽  
Vol 42 (4) ◽  
pp. 637-648 ◽  
Author(s):  
Elizabeth M. Warrendorf ◽  
David Rubinstein
Keyword(s):  
Inorganic Phosphate ◽  
Adenine Nucleotides ◽  
Lactic Dehydrogenase ◽  
Human Erythrocytes ◽  
Intracellular Level ◽  
Normal Cells ◽  
Atp Level ◽  
Atp Formation ◽  
Intracellular Phosphate ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract It has previously been possible to double the level of ATP in human erythrocytes by incubation of the cells at 37° for 10 hr with glucose and adenine. The present study describes a further increase in the ATP level and some of the possible mechanisms involved. Addition of 5 mM pyruvate to a medium containing 32 mM inorganic phosphate, glucose, and adenine elevated the level of ATP threefold during a 10-hr incubation. Pyruvate could be replaced by inosine but the presence of both limited the elevation of ATP to twice that of fresh cells. This limitation may be overcome by the use of 96 mM phosphate in the incubation medium, in which case the intracellular level of ATP is tripled within 2 hr. The conditions which limit the accumulation of ATP are associated with low intracellular phosphate concentrations and the accumulation of organic phosphates, especially, in the presence of inosine, 2,3-diphosphoglycerate. Utilizing 14C-glucose labeled in carbons 1, 2, or 6, it has been shown that when ATP is being rapidly elevated, the pentose moiety of the adenine nucleotides is mainly supplied (about 80%) by oxidation of carbon 1 of glucose, catalyzed by the dehydrogenases of the hexosemonophosphate shunt. In the presence of pyruvate this activity is doubled. Pyruvate reoxidizes NADPH formed by this pathway, since lactic dehydrogenase has some specificity towards the NADPH. The involvement of the dehydrogenases of the hexosemonophosphate shunt is illustrated by the use of erythrocytes deficient in glucose-6-phosphate dehydrogenase. Incubation of these cells for 5 hr with glucose and adenine results in only a slight increase in ATP formation, and pyruvate has no additional effect. Addition of inosine, however, leads to the same increment in ATP levels seen in normal cells. The ATP and 2,3-diphosphoglycerate levels in 6-wk preserved blood can also be increased to three times that of fresh cells by incubation with glucose, adenine, pyruvate, and inosine in a medium high in inorganic phosphate.

Ischemic preconditioning attenuates postischemic leukocyte adhesion and emigration

1996 ◽  
Vol 271 (5) ◽  
pp. H2052-H2059 ◽  
Author(s):  
T. Akimitsu ◽  
D. C. Gute ◽  
R. J. Korthuis
Keyword(s):  
Adenosine Deaminase ◽  
Topical Application ◽  
Ischemic Preconditioning ◽  
Intravital Microscopy ◽  
Leukocyte Adhesion ◽  
Inhibitory Effect ◽  
Cremaster Muscle ◽  
Beneficial Effects ◽  
Reperfusion Period

Intravital microscopy was used to determine whether ischemic preconditioning (IPC; 5 min ischemia and 10 min reperfusion) would attenuate leukocyte adhesion and emigration induced by subsequent prolonged ischemia (60 min) and reperfusion (60 min) (I/R) in murine cremaster muscle and whether adenosine produced during IPC and/or reperfusion contributed to these beneficial effects. I/R elicited a marked increase in the number of adherent and emigrated leukocytes compared with the nonischemic control muscles, an effect that was largely prevented by IPC. Superfusion of the cremaster with adenosine deaminase only during IPC or only during 60-min reperfusion attenuated the inhibitory effect of IPC on postischemic leukocyte adhesion and emigration. However, the beneficial effects of IPC were mimicked in cremaster muscles preconditioned with adenosine (topical application for 10 min beginning 20 min before the onset of prolonged ischemia). Similar results were obtained in experiments in which adenosine was topically applied to the cremaster only during the 60-min reperfusion period. Our findings suggest that the ability of IPC to attenuate postischemic leukocyte adhesion and emigration may be mediated by adenosine released during IPC and during reperfusion after prolonged ischemia.

Metabolism of adenine nucleotides in the cultured fetal mouse heart

1977 ◽  
Vol 233 (2) ◽  
pp. H282-H288
Author(s):  
I. A. Kaufman ◽  
N. F. Hall ◽  
M. A. DeLuca ◽  
J. S. Ingwall ◽  
S. E. Mayer
Keyword(s):  
Organ Culture ◽  
Adenosine Deaminase ◽  
De Novo ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Mouse Heart ◽  
Nucleotide Synthesis ◽  
Fetal Mouse ◽  
Deprivation Period

Intact beating fetal mouse hearts in organ culture were deprived of oxygen and glucose for up to 4 h, resulting in loss of beating, an 80% fall in ATP, reduction of energy charge from 0.85 to 0.48, and doubling of total nucleoside concentration. Radiolabeled adenine nucleotides were degraded to hypoxanthine and inosine, which were lost from the hearts into the medium during the deprivation period. Adenosine and adenine also appeared in the medium when adenosine deaminase was inhibited. After 24 h of O2 and glucose resupply, ATP returned to 60% of control, and energy charge rose to 0.76. Labeled nucleosides and bases remaining in the heart or exogenous labeled adenine were utilized to resynthesize ATP. [14C]glycine was rapidly taken up by recovering hearts but was not used for de novo adenine nucleotide synthesis. Ability to recover ATP and spontaneous contraction appear related to residual nucleotide and nucleoside content rather than to energy charge.

Role of adenosine as an endogenous regulator of respiration in hamster brown adipocytes

1984 ◽  
Vol 246 (3) ◽  
pp. C301-C307 ◽  
Author(s):  
R. J. Schimmel ◽  
L. McCarthy
Keyword(s):  
Adenylate Cyclase ◽  
Adenosine Deaminase ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Brown Adipocytes ◽  
Adenosine Receptor Antagonist ◽  
Endogenous Regulator ◽  
Camp Generation ◽  
Adenosine Analogue

The action of endogeneous adenosine on isolated hamster brown adipocytes was examined. Adenosine production from brown adipocytes was measured after labeling of the intracellular nucleotide pool with [3H]adenine. Accumulation of [3H]adenosine in the incubation medium was maximum after 5 min of incubation and was still present after 20 min. When adenosine accumulation was prevented by addition of adenosine deaminase, the stimulatory effects of isoproterenol on oxygen uptake, lipolysis, and adenosine 3',5'-cyclic monophosphate (cAMP) generation were enhanced. However, basal rates of lipolysis and oxygen consumption and levels of cAMP were not affected on addition of adenosine deaminase. A similar potentiation of isoproterenol responses was produced by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine, present at a concentration (10 microM) which did not change basal levels of respiration or lipolysis. Addition of the adenosine analogue 2-chloroadenosine antagonized isoproterenol-stimulated respiration and lipolysis and prevented potentiation of isoproterenol responses with 3-isobutyl-1-methylxanthine. To localize the site of adenosine action, activity of adenylate cyclase in membrane preparations from brown adipocytes was measured. Isoproterenol-stimulated adenylate cyclase activity was partially inhibited by 2-chloroadenosine in a GTP-dependent manner. Addition of Na+ enhanced the inhibitory effect of 2-chloroadenosine, and 3-isobutyl-1-methylxanthine blocked it. The calculated 50% effective dose for 2-chloroadenosine inhibition was between 10 and 15 nM. These data suggest that adenosine produced by brown adipocytes is an endogenous regulator of respiration in these cells acting at the level of the adenylate cyclase enzyme.

scholarly journals Cytochemical studies of myocardial adenylate cyclase after its activation and inhibition.

1984 ◽  
Vol 32 (1) ◽  
pp. 105-113 ◽  
Author(s):  
J Slezak ◽  
S A Geller
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Adenylate Cyclase ◽  
Adenosine Deaminase ◽  
Incubation Medium ◽  
Calcium Release ◽  
Inhibitory Effect ◽  
Alkaline Phosphatases ◽  
Similar Role ◽  
Adenosine Triphosphatases ◽  
Junctional Sarcoplasmic Reticulum

Incubation medium, as previously described (J Histochem Cytochem 27:774, 1979), was used to demonstrate the presence of adenylate cyclase (AC) in myocardium. NaF and ouabain were used to inhibit adenosine triphosphatases (ATP) and NaF and isoproterenol were used as activators of AC. The inhibitory effect of adenosine on AC was blocked by the addition of adenosine deaminase. The addition of tetramisol blocked the influence of the alkaline phosphatases on adenylyl imidodiphosphate hydrolysis. The use of these substances resulted in specific precipitation localized in junctional sarcoplasmic reticulum and sarcolemma. The reaction product was dramatically intensified after activation of AC by NaF or isoproterenol. Preincubation in 10-100 mM of propranolol, for 30 min, blocked AC stimulation by isoproterenol and prevented the appearance of the specific precipitate. The localization of specific precipitate in junctional sarcoplasmic reticulum and subsarcolemmal cisternae corresponds to the localization of Na+, K+ ATPase and may reflect the similar role that AC and Na+, K+ ATPase play in calcium release from sarcoplasmic reticulum of internal and peripheral couplings.

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