A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors

We have examined the role of ATP-dependent P2X(1) receptors in megakaryocytes (MKs) and platelets using receptor-deficient mice and selective agonists. Alpha,beta-meATP- and ATP- evoked ionotropic inward currents were absent in whole-cell recordings from MKs of P2X(1)(-/-) mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X(1) receptors. P2X(1) receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. P2Y receptor-stimulated [Ca(2+)](i) responses were unaffected in MKs from P2X(1)(-/-) mice, however the inward cation current associated with Ca(2+) release was reduced by approximately 50%, suggesting an interaction between the membrane conductances activated by P2X(1) and P2Y receptors. Interaction between P2X(1) and P2Y receptors in human platelets was also examined using [Ca(2+)](i) recordings from cell suspensions. Alpha,beta-meATP (10 microM) evoked a rapid transient P2X(1) receptor-mediated increase in [Ca(2+)](i), whereas ADP-(10 microM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co-application of alpha, beta-meATP and ADP resulted in marked acceleration and amplification of the peak [Ca(2+)](i) response. We conclude that ionotropic P2X(1) receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.     

Noradrenaline and extracellular nucleotide cotransmission involves activation of vasoconstrictive P2X(1,3)- and P2Y6-like receptors in mouse perfused kidney

Nucleotides like ATP and UTP act as potent extracellular signalling molecules. Released from sympathetic nerve endings as cotransmitters of noradrenaline or paracrine from nonexcitatory cells, they activate specific receptors (ion-gated P2X(1-7) and G-protein-coupled P2Y(1,2,4,6,11-15)). Which of these subtypes, however, are able to modulate vasoconstriction in the kidney is unclear. Wild-type- and P2Y4-receptor-deficient mice kidneys were isolated and perfused with Krebs-Henseleit solution. Pressor responses to renal nerve stimulations (RNS) and added drugs were recorded. Release of endogenous noradrenaline was measured by HPLC. RNS (1-15 Hz) induced a frequency-dependent increase in the perfusion pressor (14.2+/-5.1-67.3+/-6.9 mmHg) and noradrenaline release (1.4+/-0.3-24.2+/-3.4 ng g(-1) kidney). Pressor responses to RNS were not (1-2 Hz) or only partially (5-15 Hz) blocked by the alpha-adrenoceptor antagonist phentolamine (1 microM). Combination of phentolamine and the P2-receptor blocker PPADS (5 microM) prevented RNS-induced pressor responses. The P2X(1,3)-receptor selective antagonist NF279 (10 microM) reduced RNS-induced pressor responses in a frequency-dependent manner. Perfusion of ATP, ADP, UTP, UDP and alpha,beta-meATP concentration dependently increased perfusion pressor with the following rank order of potency alpha,beta-meATP>ADP approximately ATP approximately UDP > or = UTP. NF279 (10 microM) reduced alpha,beta-meATP- (0.1 microM) (21.7+/-3.9% of control) but not UTP- (0.3 microM) (102.6+/-15.3% of control) induced pressor responses. No differences in nucleotide-induced effects were detected among wild-type and P2Y4-receptor knockout mice. Continuous perfusion of alpha,beta-meATP (0.01 microM) potentiated UTP-, UDP- and ATP-gamma S-induced pressor responses. Neuronally and paracrine-released nucleotides evoked renal vasoconstriction by activation of P2X(1,3)- and P2Y6-like receptors in mice. Pretreatment with the P2X(1,3)-receptor agonist alpha,beta-meATP potentiated P2Y6-like receptor-mediated vasoconstrictions.     

Regional variation in P2 receptor expression in the rat pulmonary arterial circulation

The P2 receptors that mediate contraction of the rat isolated small (SPA, 200-500 micro m i.d.) and large (LPA, 1-1.5 mM i.d.) intrapulmonary arteries were characterized. 2 In endothelium-denuded vessels the contractile order of potency was alpha,beta-methyleneATP (alpha,beta-meATP)>UDP=UTP=ATP=2-methylthioATP>ADP in the SPA and alpha,beta-meATP=UTP>or=UDP>2-methylthioATP, ATP>ADP in the LPA. alpha,beta-meATP, 2-methylthioATP and ATP had significantly greater effects in the SPA than the LPA (P<0.001), but there was no difference in the potency of UTP or UDP between the vessels. 3 In the SPA, P2X1 receptor desensitisation by alpha,beta-meATP (100 microM) inhibited contractions to alpha,beta-meATP (10 nM-300 microM), but not those to UTP or UDP (100 nM-300 microM). In the LPA, prolonged exposure to alpha,beta-meATP (100 microM) did not desensitize P2X receptors. 4 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and reactive blue 2 (RB2) (30-300 microM) inhibited contractions evoked by alpha,beta-meATP. UTP and UDP were potentiated by PPADS, unaffected by RB2 and inhibited, but not abolished by suramin. 1 and 3 mM suramin produced no further inhibition, indicating suramin-resistant components in the responses to UTP and UDP. 5 Thus, both P2X and P2Y receptors mediate contraction of rat large and small intrapulmonary arteries. P2Y agonist potency and sensitivity to antagonists were similar in small and large vessels, but P2X agonists were more potent in small arteries. This indicates differential expression of P2X, but not P2Y receptors along the pulmonary arterial tree.     

The platelet ATP and ADP receptors

Adenine nucleotides, ADP and ATP, are coreleased from dense granules during platelet activation, as well as from endothelial cells and damaged red blood cells following vascular injury. Through autocrine and paracrine mechanisms, these extracellular signaling molecules interact with the platelet P2 receptors to amplify ongoing platelet activation. Two receptors for ADP, the G(q)-protein-coupled P2Y1 and G(i)-protein-coupled P2Y12 and one receptor for ATP, the P2X1 ion channel, have been identified on platelets. Due to distinct pharmacological properties and differential regulation, the P2Y and P2X receptors essentially operate on different scales of time and distance and trigger selective intracellular signaling cascades. Recent advances in the understanding of the P2Y receptor physiology have reinforced the concept of these receptors as useful targets for antithrombotic therapy. The function of P2X1 in platelet activation only recently started to be unraveled. This review focuses on recent findings on the physiology of these platelet ADP and ATP receptors, their distinct downstream intracellular signaling pathways as well as on the available agonists, antagonists and inhibitors that allow their pharmacological discrimination.     

Inhibition of adenylyl cyclase by neuronal P2Y receptors

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 - 100 microM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by ADPbetaS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATP(alpha)S, and 2-methylthio-AMP. RT - PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.     

Activation of adenosine and P2Y receptors by ATP in human peripheral nerve

Receptors for ATP in the peripheral nervous system may contribute to the transduction of sensory, including nociceptive, stimuli and are candidates in the pathogenesis of neuropathic pain. In a complex neural tissue, such as the human peripheral nerve trunk, ATP may activate P2X, P2Y, and adenosine receptors present on various cell types. Experiments were performed on segments of isolated human sural nerves. The experimental set-up enabled simultaneous recording of C fiber excitability, intracellular Ca(2+) ([Ca(2+)](i)) and extracellular K(+) activity (aK(e)). The increase in excitability of unmyelinated fibers seen during bath application of both ATP and adenosine was reversed to a reduction in axonal excitability in the presence of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolol[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an antagonist of adenosine A2 receptors. The pharmacological profile of the axonal subexcitability indicates the presence and activation of adenosine A1 receptors. Intracellular Ca(2+) transients were observed during bath application of ATP but not of adenosine and were blocked by 2'-deoxy- N(6)-methyladenosine 3',5'-bisphosphate (MRS 2179), an antagonist at P2Y(1) receptors. K(+)-sensitive microelectrodes were used to search for a possible activation of P2X receptors by ATP. In isolated rat vagus nerve, activation of P2X receptors by alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-meATP) and by diadenosine pentaphosphate (Ap5A) resulted in a rapid, transient rise in the extracellular K(+) activity. In contrast, in human nerve, application of P2X receptor agonists did not result in a detectable elevation of aK(e). The data suggest that ATP-induced changes in axonal excitability and of [Ca(2+)](i) result from activation of adenosine A2, A1 and P2Y nucleotide receptors in human nerve; a contribution of P2X receptors was not found with the methods used. It is suggested that antagonists of A2 receptors might suppress enhanced activity in human nociceptive afferent nerve fibers under conditions in which ATP and/or adenosine is released into the trunk of a human peripheral nerve.     

Diadenosine polyphosphates as antagonists of the endogenous P2Y(1) receptor in rat brain capillary endothelial cells of the B7 and B10 clones

1. Diadenosine polyphosphates (Ap(n)As, n=2 - 7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of Ap(n)As in clones of rat brain capillary endothelial cells that express P2Y(1) receptors (B10 cells) or both P2Y(1) and P2Y(2) receptors (B7 cells). 2. B10 cells responded to Ap(3)A with rises in intracellular Ca(2+) concentration ([Ca(2+)](i)). This response was prevented by adenosine-3'-phosphate-5'-phosphate, an antagonist of P2Y(1) receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP. 3. Ap(n)As inhibited ADP induced increases in [Ca(2+)](i) mediated by P2Y(1) receptors by shifting ADP concentration-response curves to larger concentrations. Apparent K(i) values were estimated to be 6 microM for Ap(4)A, 10 microM for Ap(5)A and 47 microM for Ap(6)A. Ap(2)A and Ap(3)A were much less active. 4. Ap(n)As were neither agonists nor antagonists of the endogenous P2Y(2) receptor in B7 cells. 5. Ap(n)As are neither agonists nor antagonists of the G(i)-coupled, ADP receptor in B10 cells. 6. The results suggest that most actions of Ap(n)As in B7 and B10 cells can be accounted for by endogenous P2Y(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A are specific antagonists of endogenous Ca(2+)-coupled P2Y(1) receptors.     

Apparent efficacy of kappa-opioid receptor ligands on serum prolactin levels in rhesus monkeys

ADP produces a series of responses in rabbit platelets such as shape changes, aggregation and intracellular Ca2+ mobilization. In human platelets, the P2X1 receptor mediates a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) upon stimulation with ADP. We investigated whether this phenomenon is also present in rabbit platelets. We found that the P2X1 receptor-mediated response was absent because there was (1) no elevation of [Ca2+]i in response to alpha,beta-methylene-ATP, a selective P2X1 receptor agonist, in fura-2-loaded platelets; (2) no change in the ADP-induced [Ca2+]i increase and platelet aggregation after P2X1 receptor desensitization with alpha,beta-methylene-ATP; (3) complete inhibition of the ADP-induced [Ca2+]i elevation by the P2Y1 receptor specific antagonist, A3'P5'PS, with a similar ID50 value both in the presence and absence of external Ca2+. These results indicate that ADP-induced [Ca2+]i elevation is mainly mediated by P2Y1 receptors in rabbit platelets. We conclude that the P2X1 receptor does not play a significant role in the ADP-induced platelet shape changes and aggregation.     

On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP

Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2-(methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor-selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner.     

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.     

Differential regulation and relocalization of the platelet P2Y receptors after activation: a way to avoid loss of hemostatic properties?

In the present study, we investigated the desensitization and trafficking of the P2Y1 and P2Y12 receptors after agonist-induced stimulation of platelets or astrocytoma cells transfected with the P2Y1 or P2Y12 receptors fused to green fluorescent protein. In platelets and in transfected cells, exposure to 10 microM ADP caused desensitization of the P2Y1 receptor-driven calcium signal, whereas the P2Y12 receptor-mediated inhibition of cAMP formation was not affected. Plasma membranes from ADP-stimulated platelets also retained P2Y12 activity. Agonist-induced P2Y1 receptor desensitization was accompanied by its internalization in platelets and transfected cells. In contrast, although a substantial fraction of P2Y12 receptors was rapidly and transiently internalized, most of the P2Y12 receptors remained at the plasma membrane. Activated P2Y1 receptors were internalized through a clathrin-dependent pathway in cells and platelets, whereas the P2Y12 receptors seemed to use a distinct, clathrin-independent pathway. Together, these data indicate that the P2Y1 and P2Y12 receptors are differentially regulated upon activation. The absence of desensitization of the Gi protein-coupled P2Y12 receptor-dependent responses could represent a mechanism to preserve the hemostatic properties of otherwise unresponsive platelets.     

P2 receptors in the murine gastrointestinal tract

The actions of adenosine, adenosine 5'-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1) and P2X(2) receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y(1) mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP>/=2-MeSATP>alpha,beta-meATP>ATP=UTP=adenosine. This is suggestive of P2Y(1)-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to alpha,beta-meATP. As ATP and UTP are equipotent, the presence of a P2Y(2) receptor is indicated. ATP responses were inhibited by the P2Y(1)-selective antagonist MRS 2179, and suramin. P2Y(1) receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y(1) mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N(omega)-nitro-L-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>alpha,betameATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X(2) receptors. Immunoreactivity to P2X(2) receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y(1) receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y(1), P2Y(2) and a novel P2Y receptor subtype responsive to alpha,beta-meATP in smooth muscle, while P2X(2) receptors mediate contraction of colonic smooth muscle.     

Ca(2+) signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells

1. Human embryonic kidney (HEK)-293 cells expressing recombinant G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2. Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca(2+) signalling component mediated by IL-8 (E(max)=83 +/- 8% of maximal ATP response, pEC(50) of IL-8 response=9.7 +/- 0.1). 3. 1 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects of UTP (E(max) of IL-8 response increased to 50 +/- 1% of the maximal response to ATP, pEC(50) increased to 9.8 +/- 0.1) were greater than those of 2MeSADP (E(max) increased to 36 +/- 2%, pEC(50) increased to 8.7 +/- 0.2). 5. 5. The potentiation of IL-8-mediated Ca(2+) signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca(2+) signalling was apparent in the absence of extracellular Ca(2+), demonstrating the release of Ca(2+) from intracellular stores. 6. Activation of P2Y1 and P2Y2 receptors also revealed Ca(2+) signalling by an endogenously expressed, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.     

P2Y(1) receptor activation enhances the rate of rat pinealocyte-induced extracellular acidification via a calcium-dependent mechanism

Pineal gland G-protein coupled P2Y(1) receptors potentiate noradrenaline-induced N'-acetylserotonin production, a long term response which occurs after 5 h incubation. In the current study we show that a short-term effect of stimulation of P2Y(1) receptors is the increase in extracellular acidification rate (ECAR), which is mediated by an increase in intracellular calcium concentration ([Ca(2+)](i)). The pD(2) values for ATP (3.06 +/- 0.12)-induced ECAR increase was significantly smaller (p < 0.01) than that for ADP (3.64 +/- 0.18), 2MeSATP (3.56 +/- 0.02) and 2MeSADP (3.65 +/- 0.13). The selective P2Y(1) receptor antagonists A3'P-5'P and A3'P-5'PS inhibited the increase in ECAR-induced by ADP. Clamping [Ca(2+)](i) with BAPTA (30 and 50 micromol/l) led to inhibition of ADP-induced increase in ECAR. Agonist and antagonist data indicate P2Y(1) activation leads to a [Ca(2+)](i)-dependent acidification of the extracellular medium.     

Stimulation of P2 purinergic receptors induces the release of eosinophil cationic protein and interleukin-8 from human eosinophils

1. Extracellular nucleotides are the focus of increasing attention for their role as extracellular mediators since they are released into the extracellular environment in a regulated manner and/or as a consequence of cell damage. 2. Here, we show that human eosinophils stimulated with different nucleotides release eosinophil cationic protein (ECP) and the chemokine interleukin 8 (IL-8), and that release of these two proteins has a different nucleotide requirement. 3. Release of ECP was triggered in a dose-dependent manner by ATP, UTP and UDP, but not by 2'-&3'-o-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP), ADP and alpha,beta-methylene adenosine 5' triphosphate (alpha,beta-meATP). Release of IL-8 was triggered by UDP, ATP, alpha,beta-meATP and BzATP, but not by UTP or ADP. Pretreatment with pertussis toxin abrogated nucleotide-stimulated ECP but not IL-8 release. 4. Release of IL-8 stimulated by BzATP was fully blocked by the P2X(7) blocker KN-62, while release triggered by ATP was only partially inhibited. IL-8 secretion due to UDP was fully insensitive to KN-62 inhibition. 5. Priming of eosinophils with GM-CSF increased IL-8 secretion irrespectively of the nucleotide used as a stimulant. 6. It is concluded that extracellular nucleotides trigger secretion of ECP by stimulating a receptor of the P2Y subfamily (possibly P2Y(2)), while, on the contrary, nucleotide-stimulated secretion of IL-8 can be due to activation of both P2Y (P2Y(6)) and P2X (P2X(1) and P2X(7)) receptors.