Transport and metabolism of adenosine in human blood platelets

Sixma, Jan J.; Lips, Joost P.M.; Trieschnigg, A. M. C.; Holmsen, Holm

doi:10.1016/0005-2736(76)90489-2

Cited by 39 publications

(12 citation statements)

References 25 publications

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“…In platelet suspensions hypoxanthine is almost entirely extracellular [14] and can be taken up and further metabolized via the salvage pathway [15]. Using the uptake kinetics of normal platelets [15] and similar calculations as those used for [14C]ATP (Table I), reuptake of hypoxanthine in our studies would label less than 2% of the metabolic pool in 1 h. The decrease in [14C]hypoxanthineinosine, for about 80% consisting of hypoxanthine [14], suggests that hypoxanthine incorporation can be about ten times faster in conditions of a low adenine nucleotide pool and a normal glycolytic flux.…”

Section: Discussionmentioning

confidence: 99%

Relation between energy production and adenine nucleotide metabolism in human blood platelets

Akkerman¹,

Gorter²

1980

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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SummaryThe relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN--containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN-) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 pmol • min-' • 10 -'~ cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP • equiv.-min-'-10 -'~ cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.

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Section: Discussionmentioning

confidence: 99%

Relation between energy production and adenine nucleotide metabolism in human blood platelets

Akkerman¹,

Gorter²

1980

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…Now adenosine as well as activat-cAMP in Inhibition ofHurnan and Rat Platelets 37 ing adenylate cyclase in the platelet membrane is also taken up into the platelets. The uptake proceeds by two different systems (Rozenberg & Holmsen, 1968;Sixma et al, 1976). The rapid uptake of adenosine through the platelet membrane could limit its concentration at the receptors on the membrane responsible for activating adenylate cyclase, a suggestion analogous to that put forward to explain the ineffectiveness of tryptamine on membrane receptors of smooth muscle when compared with that of 5-hydroxytryptamine (Handschumacher & Vane, 1967).…”

Section: Discussionmentioning

confidence: 99%

Relation between the Inhibition of Aggregation and the Concentration of cAMP in Human and Rat Platelets

et al. 1976

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Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.

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“…A sample of the extract was subjected to high voltage electrophoresis and radioactivity of metabolites was determined as described previously [5,21]. In a few experi-ments ADP binding to intact platelets was determined with the centrifugation technique used by Born and Feinberg [9] with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

“…The appearance of [~4C]ATP (and [~4C]AMP) may indicate that part of the accumulated radioactivity is taken up after extracellular degradation of ADP to adenosine. Adenosine is transported into the platelet and metabolized to adenine nucleotides [5]. This possibility was ruled out in a binding study in which 1 mM of unlabeled adenosine was added.…”

Section: Adp Binding To Intact Plateletsmentioning

confidence: 99%

“…It was demonstrated that ADP is not taken up into the platelets [2,3]. In platelet rich plasma ADP is degraded to adenosine and this is transported and converted intracellularly into adenine nucleotides [4,5]. The uptake of adenosine did not correlate with aggregation [6], and adenosine appeared to be an inhibitor of ADP induced aggregation [7].…”

Section: Introductionmentioning

confidence: 99%

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Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

Lips¹,

Sixma²,

Schiphorst³

1980

Biochimica et Biophysica Acta (BBA) - General Subjects

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SummaryThe equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 pM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets.The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8.A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 gM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Ql0 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism.All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.

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Transport and metabolism of adenosine in human blood platelets

Cited by 39 publications

References 25 publications

Relation between energy production and adenine nucleotide metabolism in human blood platelets

Relation between energy production and adenine nucleotide metabolism in human blood platelets

Relation between the Inhibition of Aggregation and the Concentration of cAMP in Human and Rat Platelets

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

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