Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture

Pearson, Jeremy D.; Carleton, J S; Gordon, John L.

doi:10.1042/bj1900421

Cited by 317 publications

(131 citation statements)

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“…Endothelial cells line the inner blood vessel walls and contain various ectonucleotidase activities [22]. Very recently Goldman and coworkers have shown that intact porcine aortic endothelial cells can efficiently hydrolyze extracellular Ap3A.…”

Section: Discussionmentioning

confidence: 99%

Catabolism of Ap₄A and Ap₃A in whole blood

Lüthje

Ogilvie

1988

European Journal of Biochemistry

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Adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets. After activation of the platelets both dinucleotides are released into the extracellular milieu where they play a role in the modulation of platelet aggregation and also in the regulation of the vasotone. It has recently been shown that the dinucleotides are degraded by enzymes present in the plasma [Luthje, J. & Ogilvie, A. (1987) Eur. J . Biochem. 169, 385-3881. The further metabolism as well as the role of blood cells has not been established. The dinucleotides were first degraded by plasma phosphodiesterases yielding ATP (ADP) plus AMP as products which were then metabolized to adenosine and inosine. The nucleosides did not accumulate but were very rapidly salvaged by erythrocytes yielding intracellular ATP as the main product.Although lysates of platelets, leucocytes and red blood cells contained large amounts of Ap3A-degrading and Ap4A-degrading activities, these activities were not detectable in suspensions of intact cells suggesting the lack of dinucleotide-hydrolyzing ectoenzymes. Compared to ATP, which is rapidly degraded by ectoenzymes present on blood cells, the half-life of Ap4A was two to three times longer.Since the dinucleotides are secreted together with ADP and ATP from the platelets, we tested the influence of ATP on the rate of degradation of Ap4A. ATP at concentrations present during platelet aggregation strongly inhibited the degradation of Ap,A in whole blood. It is suggested that in vivo the dinucleotides are protected from degradation immediately after their release. They may thus survive for rather long times and may act as signals even at sites far away from the platelet aggregate.Adenosine(5')tetraphospho(5')adenosine (Ap,A) and the homologue adenosine( 5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets [l, 21. Both dinucleotides are released from the platelet dense granules into the extracellular milieu after activation of the cells [l, 21. Ap3A and Ap4A have been suggested to play a role in platelet physiology. Ap4A inhibits ADP-induced platelet aggregation in platelet-rich plasma as well as in whole blood [3, 41. In contrast, Ap3A causes a gradual aggregation of platelets. This Ap3A-induced aggregation is mediated by an enzymatic activity in plasma that hydrolyzes Ap3A, producing ADP, a potent stimulator of platelet aggregation [4]. Furthermore, previous results have shown that Ap4A and Ap3A not only modulate platelet aggregation but also have vasomotor effects [5]. Thus, increasing experimental evidence suggests that these dinucleotides are regulatory molecules involved in the complex process of hemostasis.With respect to their possible role as extracellular signals it is of interest to know how long the dinucleotides can survive Enzymes. The enzymes in the plasma splitting Ap,A and Ap4A are 5'-nucleotide phosphodiesterases (EC 3.1.4.1); lactate dehydrogenase (EC 1.1.1.27).in the blood stream. After their releas...

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Section: Discussionmentioning

confidence: 99%

Catabolism of Ap₄A and Ap₃A in whole blood

Lüthje

Ogilvie

1988

European Journal of Biochemistry

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“…PGI 2 and NO are constitutively released by endothelial cells but their synthesis is increased in response to a similar range of agonists, notably molecules involved in the coagulation process (e.g., bradykinine and thrombin) or secreted by aggregating platelets (e.g., ATP) and thus serve to limit the intravascular extent of forming thrombi. In addition to NO and PGI 2 , endothelium also displays ectonucleotidases at its luminal surface (Pearson et al, 1980). These enzymes hydrolyse ATP and ADP, both potent platelet aggregating agents, into AMP and adenosine.…”

Section: Coagulation and Fibrinolysismentioning

confidence: 99%

Endothelial cell functions

Michiels

2003

Journal Cellular Physiology

612

429

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Endothelial cells play a wide variety of critical roles in the control of vascular function. Indeed, since the early 1980s, the accumulating knowledge of the endothelial cell structure as well as of the functional properties of the endothelial cells shifted their role from a passive membrane or barrier to a complex tissue with complex functions adaptable to needs specific in time and location. Hence, it participates to all aspects of the vascular homeostasis but also to physiological or pathological processes like thrombosis, inflammation, or vascular wall remodeling. Some of the most important endothelial functions will be described in the following review and more specifically, their role in blood vessel formation, in coagulation and fibribolysis, in the regulation of vascular tone as well as their participation in inflammatory reactions and in tumor neoangiogenesis. J. Cell. Physiol. 196: 430–443, 2003. © 2003 Wiley‐Liss, Inc.

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“…[4]). Assays were therefore performed at a fixed substrate concentration (100 pM; below the K , for ATP or ADP catabolism [6]) chosen to achieve adequate catabolism in reasonably short times. When the ectodiphosphatase was provided with Fig.…”

Section: Resultsmentioning

confidence: 99%

Investigation of the preferred Mg(II)‐adenine‐nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

Pearson

Cusack

1985

European Journal of Biochemistry

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In the presence of Mg' + the ecto-(nucleoside diphosphatase) on intact vascular endothelial or smooth muscle cells in culture selectively catabolises the PS diastereoisomer of adenosine 5'-[~-thio]diphosphate, (PSI-ADP [aS], and the ecto-(nucleoside triphosphatase) selectively catabolises the PS isomer of adenosine 5'- [P-thio] [l]) have proved to be powerful tools with which to analyse the stereochemical course of phosphorylation of phosphohydrolase reactions. The presence of stereoselectivity towards one isomer makes it likely that the enzyme under investigation recognises an Mg-nucleotide complex at its active site, selectivity being imposed because Mg2+ preferentially binds to oxygen rather than to sulphur in the analogues. This can be demonstrated unequivocally, and the conformation of the Mg(I1)-nucleotide complex involved at the active site assigned, by comparing the observed stereoselectivity in the presence of different bivalent metal cations with known preferences for co-ordination to oxygen or sulphur ligands. Previous studies of this type have used purified enzymes, usually kinases (e. g. [2 -51). In our study of the characteristics of ectonucleotidases on vascular endothelial and smooth muscle cells we found that each cell type possesses distinct nucleoside triphosphatase, nucleoside diphosphatase and 5'-nucleotidase ectoenzymes whose activity can be assayed using intact cells in culture. We also noted that catabolism of ATP [pS] Enzymes. Nucleoside triphosphatase (EC 3.6.1.15); nucleoside diphosphatasc (EC 3.6.1.6); 5'-nucleotidase (EC 3.1.3.5). MATERIALS AND METHODS Cell cultureEndothelial and smooth muscle cells were separately isolated from the aortas of newborn pigs and cultured as previously described [9, 101. Cells were used for experiments after 2 -17 passages. They were grown to confluence on the surface of 16-mm-diameter wells of 24-well tissue-culture trays (Nunc) and used after briefly rinsing twice with bivalentcation-free phosphate-buffered saline. Replicate incubations at 37°C were then carried out in the same saline containing the appropriate phosphorothioate analogue (100 pM) and 0.8 mM M g Z f , CO" or Cd' '. Subsamples of the incubation solutions were removed at timed intervals and stored at -20 "C until required. Analogues and measurement of metabolitesThe PR and PS diastereoisomers of ADP [aS], ATP [aS] and ATP [pS] were synthesised and purified as previously described [l, 7, 111. Catabolism of each isomer was assessed directly by HPLC separation of non-extracted subsamples of the incubation solutions, using reversed-phase HPLC [7]. Rates of catabolism were measured by analysis of the peak heights or areas on the HPLC recordings. RESULTSEssentially similar results were found for both endothelial cells and smooth muscle cells; we have therefore presented data for endothelial cells alone. Since we were working with limited numbers of intact cells, rather than purified enzymes, we could not practicably determine K , and V,,, values for each isomer (both of which are likely to ...

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Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture

Cited by 317 publications

References 37 publications

Catabolism of Ap₄A and Ap₃A in whole blood

Catabolism of Ap₄A and Ap₃A in whole blood

Endothelial cell functions

Investigation of the preferred Mg(II)‐adenine‐nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

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Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture

Cited by 317 publications

References 37 publications

Catabolism of Ap4A and Ap3A in whole blood

Catabolism of Ap4A and Ap3A in whole blood

Endothelial cell functions

Investigation of the preferred Mg(II)‐adenine‐nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

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Catabolism of Ap₄A and Ap₃A in whole blood

Catabolism of Ap₄A and Ap₃A in whole blood