Gennady G Yegutkin (1) & José González-Alonso (2)

1: MediCity Research Laboratory, University of Turku and National Public Health Institute, Turku, Finland
2: Centre for Sports Medicine and Human Performance, Brunel University, Uxbridge, UK

https://doi.org/10.36866/pn.68.31

Extracellular ATP and other nucleotides (ADP, UTP, UDP) are important signalling molecules in the cardiovascular system, where they induce diverse vasodilatatory, immunomodulatory and prothrombotic responses (Bours et al. 2006; Burnstock, 2006). These effects are mediated through G-protein-coupled P2Y receptors as well as via ligand-gated P2X receptors (Fig.1A), which are ubiquitously expressed on various cell types, including the vascular endothelium and haematopoietic cells. Subsequent to signal transduction, nucleotides need to be rapidly inactivated and vascular endothelial ectoenzymes nucleoside triphosphate diphosphohydrolase (NTPDase; known as ecto-ATPDase, CD39) and ecto-5’-nucleotidase/CD73 are considered the major regulators of the duration and magnitude of purinergic signalling in the vasculature (Bours et al. 2006). In contrast to traditional paradigms that focus on nucleotide-inactivating mechanisms, it has now become clear that ‘classical’ intracellular enzymes, adenylate kinase and nucleoside diphosphate kinase, are also co-expressed on surfaces of endothelial cells, lymphocytes and other cell types and finely control local nucleotide concentrations via backward ATP-regenerating pathway (Fig. 1B) (Yegutkin et al. 2002). The generated adenosine, in turn, has a non-redundant counteracting role in the attenuation of inflammation and mediates cardioprotective, vasodilatory, angiogenic and other responses via interaction with own G-protein-coupled receptors (Bours et al. 2006). Extracellular adenosine is then either transported into the cell by nucleoside transporters or further inactivated to inosine via ecto-adenosine deaminase reaction (Yegutkin et al. 2002). Together, the extracellular nucleotide turnover depends on functional interactions between distinct processes including:

• transient release of ATP, ADP and other agonists;
• triggering of signaling events via nucleotide- and nucleoside-selective receptors;
• ectoenzymatic inactivation;
• nucleoside uptake by the cell.

The concept of a purinergic signalling system is now widely appreciated, and studies on pathophysiology and therapeutic potential of extracellular purines represent a novel and rapidly expanding field (Burnstock, 2006). In particular, recent findings provide evidence for important roles of circulating nucleotides in the regulation of platelet reactivity, haemostasis and blood flow under exercising conditions. Regular exercise training is consistently associated with a variety of favourable alterations in cardio-vascular function, including reduced heart rate and increased maximal oxygen uptake, reduced blood pressure, activation of fibrinolysis and lower platelet activation. However, unfavourable haemostatic changes might occur at extreme exercise and environmental conditions that predispose to occlusive thrombus formation in coronary or cerebral vessels, and the extremely rare phenomenon of sudden cardiac death during exertion. Platelet activation and recruitment, followed by haemostatic plug formation, is generally initiated either via formation of thromboxane-A2 by cyclooxygenase or secretion of ADP from dense granules with subsequent activation of platelet ADP-selective P2Y1/P2Y12 receptors. In turn, vascular endothelium controls platelet reactivity and prevents thrombus formation via three pathways, including nitric oxide and prostaglandin-I2 synthesis and ADP scavenging via NTPDase activity (Burnstock, 2006).

Recently, we have shown that intravascular nucleotide turnover is acutely activated both in endurance-trained and sedentary subjects during performance of maximal cycling exercise (Yegutkin et al. 2007). A salient finding of this work is the demonstration that plasma from exercising humans, but not from resting control samples, up-regulates the expression level of the platelet activation marker P-selectin and that these prothrombotic effects can be attenuated after scavenging nucleotides by exogenous apyrase (Fig. 2). Subsequent reverse-phase high-performance liquid chromatographic analysis directly confirmed a significant increase in plasma ADP during exercise. This work additionally pointed to a role of ADP in platelet function beyond its immediate activity as a primary agonist. Probably, other synergistic factors like adrenaline (epinephrine) or some chemokines are released simultaneously with ADP that, in conjunction with the increased blood flow, would provide the stimuli for platelet activation during exercise.

While preferential activation of the coagulation cascade may predispose exercising subjects to the enhanced risk of intravascular thrombosis formation, other accompanying haemostatic changes such as activation of fibrinolysis and increased blood flow should work to counterbalance it. Blood flow and its surrogate oxygen delivery regulation are generally thought to result from the interplay of neural, myogenic and metabolic signals. A number of observations raised the possibility that purinergic signalling can also be implicated in the precise regulation of oxygen supply to contracting muscle under exercising and other hypoxic and hypercapnic conditions. Specifically, in addition to serving as an efficient oxygen carrier, the red blood cells act as sensors and controllers of local blood flow via transient release of ATP in proportion to the degree of haemoglobin deoxygenation (González-Alonso et al. 2002; Ellsworth, 2004). The released ATP subsequently induces a conducted vasodilatatory response upstream and regulates oxygen supply to contracting muscles via binding to the endothelial P2Y1/P2Y2 receptors and stimulation of vascular endothelium to release nitric oxide and arachidonic acid metabolites (Ellsworth, 2004; Burnstock, 2006).

Identification of a network of soluble purine-converting enzymes freely circulating in the bloodstream adds another level of complexity to understanding the regulatory mechanisms of purine homeostasis within the vasculature. We have shown that two soluble nucleotide-inactivating enzymes, nucleotide pyrophosphatase/ phospho-diesterase (NPP) and NTPDase, constitutively circulate in the human bloodstream, and we have further demonstrated that their activities are transiently up-regulated during strenuous exercise by 20–25 and 80–100%, respectively (Yegutkin et al. 2007). The exercise-mediated increase revealed in serum NPP activity may allow the by-passing of the generation of a principal platelet-recruiting agent ADP, via direct conversion of circulating ATP into AMP and PPi. Furthermore, concurrent activation of another soluble nucleotidase NTPDase might represent a novel and currently unappreciated effector system contributing, along with vascular endothelial NTPDases, to the termination of acute prothrombotic effects of ADP under hyperaemic exercising conditions. Of note is the fact that the recombinant soluble form of human NTPDase/CD39 is currently considered a promising aspirin-insensitive antithrombotic drug, which potently inhibits platelet reactivity under various experimental prothrombotic conditions. Therefore, data on constitutive presence of soluble NTPDase in human blood and its up-regulation during exhaustive exercise may open up further research for future therapeutic applications of this major ADP-inactivating nucleotidase as a ‘natural antithrombotic enzyme’ for anti-platelet therapy in hypoxia-associated and other vascular diseases.

In summary, transient exercise-mediated increases in circulating ATP and ADP levels, together with concurrent up-regulation of soluble nucleotide-inactivating activities induced by endurance training, may represent an efficient control system that finely regulates both tissue O2 delivery and platelet reactivity in healthy subjects. On the other hand, acute disturbances in the pattern of intravascular nucleotide turnover occurring during exhaustive exertion might contribute, in conjunction with other prothrombotic synergistic factors, to the enhanced risk of cardiovascular morbidity and mortality, especially in the elderly and sedentary subjects suffering from endothelial dysfunction and insufficient release of anti-platelet compounds.

References

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Yegutkin GG, Samburski SS, Mortensen SP, Jalkanen S & González-Alonso J (2007). Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans. J Physiol 579, 553–564.