Blood cells: an historical account of the roles of purinergic signalling.

The involvement of purinergic signalling in the physiology of erythrocytes, platelets and leukocytes was recognised early. The release of ATP and the expression of purinoceptors and ectonucleotidases on erythrocytes in health and disease are reviewed. The release of ATP and ADP from platelets and the expression and roles of P1, P2Y(1), P2Y(12) and P2X1 receptors on platelets are described. P2Y(1) and P2X(1) receptors mediate changes in platelet shape, while P2Y(12) receptors mediate platelet aggregation. The changes in the role of purinergic signalling in a variety of disease conditions are considered. The successful use of P2Y(12) receptor antagonists, such as clopidogrel and ticagrelor, for the treatment of thrombosis, myocardial infarction and stroke is discussed.

Control of coronary blood flow during exercise.

During exercise, coronary blood flow increases to match the augmented myocardial oxygen demand because of tachycardia. Coronary vasodilation during exercise is via a combination of feedforward and feedback control mechanisms. Feedforward control is mediated by sympathetic ß-adrenoceptor vasodilation. Feedback vasodilator control is via a novel hypothesis where adenine nucleotides released from red blood cells act on endothelial purinergic receptors.

Rapid resensitization of purinergic receptor function in human platelets.

BACKGROUND: Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. OBJECTIVES: To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. METHODS: These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. RESULTS: In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. CONCLUSIONS: This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

Purinergic receptors involved in the immunomodulatory effects of ATP in human blood.

We recently showed that the physiological compound ATP simultaneously inhibited TNF-alpha and stimulated IL-10 release in LPS-PHA stimulated blood. The purpose of the present study was to determine the mechanism involved in the concerted modulatory effect of ATP on TNF-alpha and IL-10. Incubation of blood with ATP in the presence of selective P2 receptor antagonists showed that the stimulatory effect of ATP on IL-10 release was completely annihilated by both 2-MeSAMP (a P2Y12/13 receptor antagonist) and PSB-0413 (a P2Y12 receptor antagonist). On the other hand, the inhibitory effect of ATP on TNF-alpha release was completely reversed by 5'-AMPS (a P2Y11 receptor antagonist) as well as by H-89, an inhibitor of cAMP-activated PKA. The concerted inhibition by ATP of TNF-alpha release via P2Y11 activation and stimulation of IL-10 release via P2Y12 activation implicates a novel approach towards immunomodulation by altering the balance among pro- and anti-inflammatory cytokines.

Evidence that most high-affinity ATP binding sites on aortic endothelial cells and membranes do not correspond to P2 receptors.

It has recently been demonstrated that two types of ATP receptors, the P2Y and P2U receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P2Y and P2U subtypes on intact bovine aortic endothelial cells and on membranes prepared from these cells using adenosine 5'-0-(3-thio[35S]triphosphate) ([35S]ATP gamma S), [alpha-32P]ATP and [alpha-32P]UTP as radioligands. [35S]ATP gamma S binding to bovine aortic endothelial cell membranes was saturable and apparently involved a single class of high-affinity binding sites (Kd: 14 +/- 11 nM. Bmax 1.6 +/- 0.7 pmol/mg protein; mean +/- S.D.). A similar class of high-affinity binding sites was identified with [alpha-32P]ATP (Kd: 14 +/- 9 nM; Bmax: 1.7 +/- 1.1 pmol/mg protein; mean +/- S.D.). Competition experiments showed that only one third of these sites bound 2-methylthio-ATP (2-MeSATP) with high affinity (Ki: 21 +/- 5 and 14 +/- 10 nM, mean +/- S.D., for [35S]ATP gamma S and [alpha-32P]ATP, respectively) and might therefore represent the P2Y receptors. UTP did not compete with [35S]ATP gamma S or [alpha-32P]ATP for binding at the remaining sites, indicating that they are not the P2U receptors. No high-affinity UTP binding sites could be detected using [alpha-32P]UTP. [35S]ATP gamma S binding to intact bovine aortic endothelial cells was competed by ATP gamma S (Kd: 1.0 +/- 0.5 microM; mean +/- S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P2Y nor the P2U receptors.

Aggregin: a platelet ADP receptor that mediates activation.

ADP is known to induce platelet shape change, aggregation, and exposure of fibrinogen binding sites as well as inhibit stimulated adenylate cyclase. The platelet is unique in that its purinergic receptor prefers ADP over ATP, which functions as a competitive antagonist. The affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), has been used to covalently label a single membrane protein, aggregin, on the external platelet surface with mol wt of 100 kDa. Concomitant with incorporation of FSBA, ADP-induced shape change, aggregation, and fibrinogen binding is inhibited. FSBA is also a weak agonist at short times and high concentration, which suggests that prior noncovalent binding to aggregin takes place before covalent modification. Aggregin differs from platelet glycoprotein IIIa in its physical and immunochemical properties. Aggregin is distinct from the receptor coupled to adenylate cyclase. Using FSBA as a probe, platelet aggregation by thromboxane A2 analogs and collagen was shown to be dependent on ADP but not the shape change induced by these agonists. Binding to aggregin is required for epinephrine-induced aggregation. In turn, epinephrine increases the affinity of ADP for its receptor. Thrombin at concentrations greater than 2 nM (0.2 units/ml) stimulates platelet aggregation independent of ADP, but by raising cytoplasmic Ca2+ it activates platelet calpain, which in turn cleaves aggregin. Thus aggregin, in addition to serving as the ADP receptor linked to shape change and aggregation, plays a role in fibrinogen receptor latency that is relieved entirely by ADP binding to or proteolysis of aggregin.

Phosphatidylinositol 4,5-bisphosphate hydrolysis in turkey erythrocytes is regulated by P2y purinoceptors.

When intact [3H]inositol-loaded turkey erythrocytes were stimulated with the purinergic agonist ADP, there was a rapid increase (2.5-fold after 30 sec) in the intracellular content of [3H]inositol 1,4,5-trisphosphate, followed by increases in the levels of [3H]inositol bisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate (4-fold and 5-fold, respectively, after 3 min). [3H]inositol monophosphate levels did not rise in the first 3 min of ADP stimulation but increased slowly thereafter, demonstrating that the primary response of turkey erythrocytes to purinergic stimulation is hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inositol phosphate accumulation was evoked by a P2y purinoceptor, as indicated by the rank order of potencies of a variety of purinergic agonists. 2-Methylthioadenosine 5'-triphosphate was the most potent agonist tested, with an EC50 value of 0.36 microM. High performance liquid chromatography analysis demonstrated the presence of three distinct inositol tetrakisphosphate isomers in [3H]inositol-loaded turkey erythrocytes, inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,5)P4], inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and inositol 3,4,5,6-tetrakisphosphate. Prolonged stimulation with adenosine 5'-O-(2-thiodiphosphate), a nonhydrolyzable analogue of ADP, resulted in a 60-fold increase in the level of [3H]Ins(1,3,4,5)P4, whereas a substantial rise in the [3H]Ins(1,3,4,6)P4 fraction was also seen. These results indicate that turkey erythrocytes represent a valuable model system for studies of purinoceptor function as well as fundamental aspects of cell surface receptor-regulated phosphoinositide metabolism.